이 작품 전자 일반 드 Asuntos 델에 의해 부분적으로 지원 되었다 개인 Académico, DGAPA/PAPIIT-UNAM (no를 부여 합니다. M.L 하 M.에 IA205117 IN219916 A)와 Consejo 나시오날 드 많은 y Tecnològia (CONACYT 보조금 번호 240614 메이저 리그에).

1. 식별, 격리 및 피 Vulgaris 닌 발기인의 복제
<ol>
	<li>관심사의 유전자의 발기인 순서를 식별 합니다. 일부의 게놈 데이터베이스와 분석 도구 Phytozome, 합 식물, NCBI, 등 A 콩과 발기인 P. vulgaris 닌 (PvNIN; 식물에 사용할 수 있는 Phvul.009G115800)는이 연구20에 사용 되었다.</li>
	<li>디자인 oligos 게이트웨이 또는 벡터 시스템에 따라 복제 하는 전통적인 제한 효소 사용 (그림 1A). 참고: 표준 (25-35 bp), desalted 뇌관 작업 괜 찮 아 요. 발기인과 유전자 사이의 측면 역 올리고 시퀀스는 좋습니다.</li>
	<li>갓 격리 P. vulgaris 에서 PvNIN 발기인 지역 (또는 관심사의 유전자의 발기인 지구)를 증폭 발기인 특정 올리고 세트를 사용 하 여 게놈 DNA. 높은 중 합 효소를 사용 하 여 다음 PCR 조건: 95 ° C 5 분; 30 사이클 (95 ° C 30에 대 한 s, 55 ° C 30 초, 1 분에 72 ° C); 그리고 5 분 동안 72 ° C입니다.</li>
	<li>7 x 10 cm 젤 트레이에 60 V 1 %agarose 젤에 증폭된 한 파편을 실행 하 고 해당 amplicon elute (700 bp, 그림 1B) 및 제조업체의 지침에 따라 pENTR/D-이동 벡터에 클론.</li>
	<li>2 µ L 유능한 대장균 으로 반응의 변환 lysogeny 형제 (파운드) 50mg/L 대 (Kan50)와 보완에 제조업체의 지침과 변환 혼합물의 플레이트 150 µ L를 사용 하 여 셀. 밤새 37 ° C에서 도금된 세포 성장.</li>
	<li>선택 3-6 식민지 문화 그들 파운드 매체 포함 하는 Kan50에서에서 하룻밤. 플라스 미드의 선택 방법을 사용 하 여 DNA를 분리 하 고 벡터 특정 M13 oligos를 사용 하 여 PCR에 의해 플라스 미드 분석. PCR, 플라스 미드, oligos의 오후 0 3의 사용 100 ng에 대 한 10 X PCR 버퍼, 0.5 U Taq DNA 중 합 효소, 그리고 dNTPs 10 m m.</li>
	<li>사용 하는 PCR 증폭 다음과; 3 분;에 대 한 95 ° C 30 사이클 (95 ° C 30에 대 한 s, 58 ° C 30에 대 한 s, 75 72 ° C s); 그리고 7 분에 대 한 72 ° C 1 %agarose 젤에 PCR 제품을 시각화. 올바른 삽입 324 bp 밴드 (그림 1C) 밴드 1024 혈압과 삽입 지 없이 벡터에 있는지 확인 합니다.</li>
	<li>LR 반응 항목 벡터 (pENTR/D-이동-PvNIN)와 대상 벡터 pBGWFS7.021 제조업체의 지침에 따라 상업 효소 혼합을 사용 하 여 수행 합니다.</li>
	<li>유능한 대장균 으로 LR 반응의 2 µ L 변환 파운드 100 mg/L spectinomycin (Spe100)의 보충에 제조업체의 지침과 접시 150 µ L의 변환에 설명 된 대로 표준 기술을 사용 하 여 셀. 밤새 37 ° C에서 도금된 세포 성장.</li>
	<li>5 식민지 약을 선택 하 고 Spe100를 포함 하는 파운드 매체에 하룻밤 문화. 선택 방법을 사용 하 여 DNA 플라스 미드를 분리 하 고 발기인 특정 oligos를 사용 하 여 PCR에 의해 플라스 미드 분석.</li>
	<li>1.3 단계에서 PCR 상태를 사용 합니다. 증폭을 확인 하는 1 %agarose 젤에 PCR 제품을 시각화 합니다. Amplicons는 700 혈압.</li>
	<li>긍정적인 플라스 미드 pBGWFS7.0/PvNIN::GUS-GFP (그림 1D) 삽입의 진위를 확인 하기 위해 발기인 특정 oligos와 시퀀스.</li>
	<li>그러나 A. rhizogenes 긴장 K599 (, A. rhizogenes 의 다른 변종 사용할 수 있습니다)에 플라스 미드를 변환. 다음 설정으로 1mm 베트에 electroporate 및 electrocompetent 세포의 50 µ L를 플라스 미드 준비의 2 µ L를 추가: 1.8 kV, 25 µ F, 및 200 Ω.</li>
	<li>셀에 복구 ~ SOC 매체의 500 µ L 2 h. 접시 파운드 Spe100에 28 ° C에서 흔들어와 28 ° C에서 2 일 동안 성장.</li>
	<li>1.7 단계에서 언급 한 대로 식민지 심사를 수행 하 고 단계 1.3에서 PCR 조건을 사용 하 여. 긍정적인 복제품에서 글리세롤 주식을 확인 하 고-80 ° C에 저장</li>
	<li>A. rhizogenes 문화 은닉 부정적인 컨트롤로 빈 pBGWFS7.0 벡터를 사용 합니다.</li>
</ol>
2. A. Rhizogenes 는 일반 콩의 털이 뿌리 변형
<ol>
	<li>콩 (P. vulgaris cv. 흑인 Jamapa) 씨앗 (약 20)을 살 균 하십시오 1 분 96% 에탄올의 50 ml에서 및 에탄올; 삭제 층 류 두건에 지속적인 혼합과 10 분 동안 20% 표 백제 (염소) 50 mL를 추가 합니다. 표 백제를 제거 하 고 3 번 초과 살 균 물에 씻어. 참고: 다른 P. vulgaris cultivars 털이 루트 유도 사용할 수 있습니다.</li>
	<li>브 로튼 적신 멸 균 필터 종이에 소독된 씨앗을 발 아 &#38; Dilworth (B &#38; D) 솔루션22 (표 1) 28 ° c.에 어둠 속에서 2 일 동안</li>
	<li>변환 하기 전에 하루 준비 다음.
	<ol>
		<li>A. rhizogenes 문화 (준비 단계 1.13 1.14) 이진 벡터 은닉의 150 µ L을 고체 파운드 Spe100에 streak 하룻밤 28 ° C에서 성장 하 고.</li>
		<li>B 15 mL 튜브를 채우기 &#38; D 솔루션 및 바꾸기 두 배 층이 된 알루미늄 호 일 나사 모자. B의 10 mL를 포함 하는 22 cm 유리 튜브에 이것을 조립 &#38; D 솔루션 (그림 2C) 및 고압 그것.</li>
	</ol>
	</li>
	<li>변화의 날, 층 류 두건에 다음 메 마른 재료 조립: 2.2 단계에서 씨앗, 단계 2.3.1, 2.3.2 단계에서 튜브, 살 균 두 배 증류수 (10 mL), 펫 및 피 펫 팁, 살 균 겸 문화 접시를 출 아 했다 그리고 3 mL 주사기입니다.</li>
	<li>구부러진된 200 µ L 팁을 사용 하 여 microcentrifuge 튜브로 접시에서 A. rhizogenes 세포를 긁어와 이중 살 균 물의 1 mL에서 resuspend. 마찬가지로, 벡터 제어 셀 별도로 준비 합니다.
	<ol>
		<li>Resuspend을 부드럽게 세포를 혼합. 와 동 하지 마십시오.</li>
	</ol>
	</li>
	<li>단계의 2.3.2 살 균 피 펫 팁을 사용 하 여 튜브의 알루미늄 호 일에 3mm 구멍을 확인 합니다.</li>
	<li>주사기와 2.5 단계에서 세포 (발기인 또는 벡터 제어)를 수집 하 고 약간 찌르는 바늘 팁 (그림 2G) 세균된 씨의 hypocotyls. 참고: 바늘 침투 (4-6 시간) 혈관 조직 다는 것을 확인 하 고 작은 주사 (~ 5 µ L)는 hypocotyl 주위에 다른 위치에서 상처에 셀의 삭제.</li>
	<li>신중 하 게 B를 포함 하는 15 mL 튜브의 3 m m 구멍에 삽입 된 묘 목의 뿌리를 삽입 &#38; D 솔루션. 유리 튜브 22 mL에 전체 설치를 반환 하 고 습도 유지 하기 위해 그들을 봉인.</li>
	<li>16 h 빛: 8 28 ° C 유지 성장 챔버에 튜브를 품 어 h 어두운 photoperiod. 참고: 3-4 일 후 부 화, 식물 성장 22 mL 유리 튜브의 가장자리에.</li></ol>이 단계에서 모자를 제거 하 고 그림2에서와 같이 줄기 주위 parafilm으로 가장자리를 밀봉 합니다.
	<li>5-7 일, 부상 사이트에서 굳은 살을 관찰 하 고 찾아 ~ 2 cm 털 뿌리 변환 후 10-12 일. 참고: 그것은 B를 유지 하는 것이 중요 &#38; 플라스틱 및 유리 튜브에 끊임없이 D 솔루션.</li>
	<li>2 주 후, 털 뿌리 아래 2 cm 줄기를 절단 하 여 기본 루트를 제거 하 고 살 균 질 석 포함 하는 남 비로 기본 뿌리를 이식 합니다. 성장 챔버 (16 빛: 8 시 28 ° C에서 어두운)에 식물을 유지 하 고 B와 관개 &#38; D 솔루션.</li>

3. 발기인 분석: 닌 발기인 식 일반 콩의 Rhizobial 혹
<ol>
	<li>뿌리 혹의 유도, 접종 Rhizobium tropici 또는 Rhizobium etli (일반 콩에 호환 되)는 PY 매체 (0.5 g 펩, 0.3 g 효 모 추출 물) 700 mM CaCl2 와 20 mg mL− 1 를 가진 보충의 100 ml nalidixic 산입니다. 300 rpm에서 떨고와 30 ° C 24 h에서 품 어. 참고: 어떤 특정 항생제 유전자 변형 Rhizobium경우 사용 될 수 있습니다.</li>
	<li>실 온에서 2800 x g에서 3 분 동안 원심 분리기에 셀 작은 고 10mm MgSO410 mL에 resuspend. 10mm MgSO4로 희석 하 여 세600 0.6 셀의 OD를 조정 합니다.</li>
	<li>문화 (3.2 단계에서 준비) 식물 (모두 발기인 및 벡터 제어) 털이 뿌리에 결 절 성장 유도 하 당 1-2 mL 접종 B 식물을 관개 &#38; D 솔루션 nodulation 홍보 제한 질소 (2mm 알 것3).</li>
	<li>실험 필요에 따라서 다른 시간 지점에서 털이 뿌리를 수집 합니다. 3-5 일 사이 감염 스레드 공부에 대 한 수집 샘플 게시물 접종 (dpi). 참고: 원시 결 절 및 젊은 혹 수 수에서 공부 6-9 dpi와 10-12 일 dpi, 각각. 마지막으로, 성숙 하 고 기능적인 혹 14-21 dpi, 및 senesced 혹에서 공부 될 수 있다 &#62; 28 dpi.</li>
	<li>거 스 (GFP) 리포터 유전자의 활동에 대 한 루트/작은 혹을 처리 합니다. 제퍼슨23에 의해 설명 하는 프로토콜에 따라 GUS 활동 분석. GFP 형광 현미경으로 관찰 합니다. GFP 형광 블루 아르곤 이온 레이저 자극 (488 nm), 510에서 540 내보낸된 형광을 수집 nm.</li>
</ol>

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M., K&#252;ster, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoter+of+the+Vicia+faba+L.+gene+VfEnod12+encoding+an+early+nodulin+is+active+in+cortical+cells+and+nodule+primordia+of+transgenic+hairy+roots+of+Vicia+hirsuta+as+well+as+in+the+prefixing+zone+II+of+mature+transgenic+V.+hirsuta+root+nodules.">The promoter of the Vicia faba L. gene VfEnod12 encoding an early nodulin is active in cortical cells and nodule primordia of transgenic hairy roots of Vicia hirsuta as well as in the prefixing zone II of mature transgenic V. hirsuta root nodules.</a> Plant Science. 160 (1), 67-75 (2000).</li><li>Szabados, L., Ratet, P., Grunenberg, B., de Bruijn, F. 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M., Blanco, L., Lara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentially+expressed+genes+in+mycorrhized+and+nodulated+roots+of+common+bean+are+associated+with+defense,+cell+wall+architecture,+N+metabolism,+and+P+metabolism.">Differentially expressed genes in mycorrhized and nodulated roots of common bean are associated with defense, cell wall architecture, N metabolism, and P metabolism.</a> PLoS ONE. 12 (8), e0182328 (2017).</li><li>Karimi, M., Inz&#233;, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway+vectors+for+Agrobacterium-mediated+plant+transformation.">Gateway vectors for Agrobacterium-mediated plant transformation.</a> Trends Plant Sci. 7 (5), 193-195 (2002).</li><li>Broughton, W. J., Dilworth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+leghemoglobin+synthesis+in+snake+beans.">Control of leghemoglobin synthesis in snake beans.</a> Biochem J. 125 (4), 1075-1080 (1971).</li><li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+chimeric+genes+in+plants,+the+GUS+gene+fusion+system.">Assaying chimeric genes in plants, the GUS gene fusion system.</a> Plant Mol Biol Rep. 5 (4), 387-405 (1987).</li><li>Cho, H. J., Farrand, S. K., Noel, G. R., Widholm, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+induction+of+soybean+hairy+roots+and+propagation+of+the+soybean+cyst+nematode.">High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode.</a> Planta. 210 (2), 195-204 (2000).</li><li>Deng, Y., Mao, G., Stutz, W., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Composite+Plants+in+Medicago+truncatula+used+for+Nodulation+Assays.">Generation of Composite Plants in Medicago truncatula used for Nodulation Assays.</a> J. Vis. Exp. (49), e2633 (2011).</li><li>Kumagai, H., Kouchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+expression+of+hairpin+RNA+in+Lotus+japonicus+roots+and+root+nodules.">Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules.</a> Mol Plant Microbe Interact. 16 (8), 663-668 (2003).</li><li>Okamoto, S., Yoro, E., Suzaki, T., Kawaguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hairy+Root+Transformation+in+Lotus+japonicus.">Hairy Root Transformation in Lotus japonicus.</a> Bio-protocol. 3 (12), e795 (2013).</li><li>Jacobs, T. B., Martin, G. 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저자는 공개 없다.

유전자의 기능 분석, 동안 유전자 식 패턴의 연구 vivo에서유전자의 공간과 일시적인 규칙 이해에 중요 한 역할을 하고있다. 진 식 패턴을 공부 하는 잘 알려진 방법 관심, 유전자 프로모터 영역을 복제 하는 것입니다 형광 표식 유전자 (GFP, RFP, 등) 등 β-glucuronidase 리포터 유전자를 업스트림. 여기, 우리는 루트 결 절 공생 (RNS) 특정 유전자의, RNS 동안 P. vulgaris spatio 시간적 식 패?...

발기인은 관심사의 유전자의 표현 규제 이해에 중요 한 역할을 하는 중요 한 분자 생물학적 도구. 프로모터는 상류에 있는 DNA 순서는 번역의 개시 코 돈 유전자의 순서 그리고 그들이 유전자;의 중앙 규제 정보 따라서, 그들의 올바른 주석 및 특성화는 유전자 기능을 이해 하는 중요 한. 식 패턴에 따라 공장 발기인 구성, 조직, 또는 개발 단계 관련 및 유도할 수 있는1으로 분류 됩니다. Transcriptomic 기술, 컴퓨터 모델링, 개선 및 다양 한 식물 종에 대 한 게놈 시퀀스의 수를 증가의 가용성 발기인 시퀀스2의 대규모 예측을 촉진 했습니다.
다른 한편으로, 그것은 또한 발기인 평가 도구와, 효율적으로, 신속 하 고 재현할 수 있는 유전자 변환 기술을 확립에 중요 한. 다른 모델 식물 달리 일반적인 콩 콩과 식물 (P. vulgaris) 유전자의 기능 특성의 안정적인 유전자 변환에 대 한 sp. Phaseolus 완강히 특성상 주로 장애가. 일시적인 변환 시스템 빠른 유전자 기능 분석 연구3에 대 한 대체 역할을 합니다. 콩과 공생 연구, 콩과 식물 호스트 공장 및 rhizobial 박테리아 사이의 상호 작용 발기인 연구 및 결 절 특정 유전자의 기능 분석에 대 한 가장 온순한 모델 시스템 중 하나입니다. 지금까지,이 공생에 관련 된 여러 가지 콩과 발기인 되었습니다 특징, 즉, 개자리속 truncatula PT44, SWEET115, 로터스 나무 키 클 롭 스, UBQ6, VAG17, 글리신 최대 PT5 8, Exo70J9, P. vulgaris RbohB10,11,12, TRE113, PI3K14, 토르15, 등. Cis 요소 직접 영향을 미치는 유전자 규칙. 녹음 방송 요인 ENBP1A의 초기 nodulin VfENOD12, Cis 규제 지역 (bp −692)에 한다이 얼 faba16의 결 절 primordia에서 취재 원 유전자의 표현을 용이 하 게 한다. Cis 규제 지역의 교체 (−48 bp −161) 결 절 특정 발기인의 leghemoglobin GLB3는 분리와 발기인 δ-p35S 및 δ-pNOS, 결 절 특이성의 손실 귀착되 었 다 잘리고 발기인 활동17 감소 .
이전 보고서 표시 녹음 방송 요인 닌 루트 머리 세포에 rhizobial 감염의 개시에 필요한 고도 결 절 organogenesis L. 나무18에 대 한 필수적 이다. 현재 연구에서 우리는 식별, 격리, 복제 및 일반적인 콩 털이 뿌리에 결 절 특정 발기인의 특성에 대 한 프로토콜을 설명 합니다. 이 위해 우리 P. vulgaris 의 rhizobial 공생 전용 닌 발기인을 선택 하 고 공상 기자 거 스 enhanced::GFP transcriptional 퓨전에 복제. 또한,이 프로토콜 P. vulgaris A. rhizogenes 유도 털 뿌리를 사용 하 여 유전자 변환의 신속 하 고 다양 한 시스템을 설명 합니다. 이 시스템은 변환 후 2 주 미만에 털이 뿌리를 생성합니다. 마지막으로, 우리는 거 스 얼룩에 의해 식민지 rhizobia 루트 혹에서 닌 발기인의 spatiotemporal 식 평가.
여기에 설명 된 절차는 콩과 식물 식물의 nodulation 및 mycorrhization11 의 연구 뿐만 아니라 뿌리19에서 발기인 식 패턴의 연구에 대 한 유용할 수 있습니다. 또한,이 프로토콜은 비 전문 실험실에서 사용 하기 쉬운입니다.

<table border="0" cellpadding="0" cellspacing="0" width="1125">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Primers for qRT-PCR assay</td>
			<td style="width:253px;"></td>
			<td style="width:460px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pNIN Forward</td>
			<td>CACC ATA GCT CCC CAA AAT GGT AT</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pNIN Reverse</td>
			<td style="width:253px;">CAT CTT CCT TCC ACT AAC TAA C</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M13 Forward</td>
			<td>GTA AAA CGA CGG CCA G</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M13 Reverse</td>
			<td>CAG GAA ACA GCT ATG AC</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name</td>
			<td style="width:253px;">Company</td>
			<td style="width:460px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">REAGENTS</td>
			<td style="width:253px;"></td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pENTR/D-TOPO Cloning Kit</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">K243520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway LR Clonase II Enzyme Mix&#160;</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">11791100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">pBGWFS7.0&#160;</td>
			<td style="width:253px;">Plant systems biology</td>
			<td style="width:460px;">https://gateway.psb.ugent.be/vector/show/pBGWFS7/search/index/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum Taq DNA Polymerase</td>
			<td style="width:253px;">ThermoFisher Scientific</td>
			<td style="width:460px;">10966018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">DNeasy Plant Mini Kit</td>
			<td style="width:253px;">Qiagen</td>
			<td>69104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">PureLink Quick Gel Extraction Kit</td>
			<td style="width:253px;">ThermoFisher Scientific</td>
			<td style="width:460px;">K210012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Platinum Pfx DNA Polymerase</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">11708013</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:245px;">Certified Molecular Biology Agarose</td>
			<td style="width:253px;">Bio-Rad</td>
			<td>1613102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">One Shot TOP10 Chemically Competent E. coli</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">C404006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Nacl</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tryptone</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">T7293-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Yeast extract</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">Y1625-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bacteriological agar</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">A5306-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Kanamycin sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">60615-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Spectinomycin sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td>PHR1441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethyl alcohol</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bacteriological peptone</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">P0556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Calcium chloride</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">C1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Nalidixic acid</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">N8878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Magnesium sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Gel loading solution</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">G7654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name</td>
			<td style="width:253px;">Company</td>
			<td style="width:460px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">EQUIPMENT</td>
			<td style="width:253px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Thermocycler</td>
			<td style="width:253px;">Veriti Thermal Cycler</td>
			<td style="width:460px;">4375786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Centrifuge</td>
			<td style="width:253px;">Sigma</td>
			<td style="width:460px;">Sigma 1-14K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Gel documentation unit</td>
			<td style="width:253px;">Carestream&#160;</td>
			<td style="width:460px;">Gel Logic 212 PRO</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">MaxQ SHKE6000 6000 Shaking Incubator - 115VAC</td>
			<td style="width:253px;">Thermo scientific</td>
			<td style="width:460px;">EW-51708-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Plant growth chamber</td>
			<td style="width:253px;">MRC</td>
			<td>PGI-550RH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Horizantal laminarair flow cabinate</td>
			<td style="width:253px;">Lumistell</td>
			<td style="width:460px;">LH-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Fluorescent microscope</td>
			<td style="width:253px;">Leica&#160;</td>
			<td style="width:460px;">DM4500 B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Petridish</td>
			<td style="width:253px;">sym laboratorios</td>
			<td style="width:460px;">90X15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Scalpel Blade&#160;</td>
			<td style="width:253px;">Fisher scientific</td>
			<td style="width:460px;">53223</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Falcon 15mL Conical Centrifuge Tubes</td>
			<td style="width:253px;">Fisher scientific</td>
			<td style="width:460px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">22 mL glass tubes</td>
			<td style="width:253px;">Thomas scientific</td>
			<td style="width:460px;">45048-16150</td>
			<td></td>
		</tr>
	</tbody>
</table>

plant promoter analysis: identification and characterization of root nodule specific promoter in the common bean

업스트림 시퀀스 시퀀스를 코딩 하는 유전자의 발기인 시퀀스도 불린다. 발기인의 식 패턴을 공부 하는 것은 유전자 규정과 spatiotemporal 식 패턴 대상 유전자의 이해에 매우 중요 한입니다. 다른 한편으로, 그것은 또한 발기인 평가 도구와, 효율적으로, 신속 하 고 재현할 수 있는 유전자 변환 기술을 확립에 중요 한. 이 연구에서 우리는 유전자 변형 털 뿌리에 Phaseolus vulgaris 의 rhizobial 공생 특정 결 절 개시 (NIN) 발기인의 spatiotemporal 식 패턴 조사. 식물 게놈 데이터베이스와 식별, 분석 도구를 사용 하 여 격리, 그리고 복제 공상 기자 transcriptional 퓨전에 P. vulgaris 닌 발기인 β-glucuronidase (거 스) 거 스 enhanced::GFP. 또한,이 프로토콜 P. vulgaris Agrobacterium rhizogenes 유도 털 뿌리를 사용 하 여 유전자 변환의 신속 하 고 다양 한 시스템을 설명 합니다. 이 시스템은 변환 후 10 ~ 12 일에 ≥2 cm 털 뿌리를 생성합니다. 다음으로, 우리 Rhizobium 접종 후 접종의 정기적으로 털 뿌리에 닌 발기인의 spatiotemporal 식 평가. GUS 활동에 의해 묘사 된 우리의 결과 표시 닌 발기인 nodulation의 과정에서 활동적 이었다. 함께, 현재 프로토콜 식별, 격리, 복제, 그리고 일반적인 콩 털이 뿌리에서 식물 발기인을 특성화 하는 방법을 보여 줍니다. 또한,이 프로토콜은 비 전문 실험실에서 사용 하기 쉬운입니다.

이 연구의 목적은 결 절-특정 P. vulgaris 닌 spatiotemporal 식 패턴을 평가 했다. 이렇게 하려면 700 bp 지역 상류 닌 유전자의 번역 개시 코 돈에의 선정 되었다 고 그림 1A에서처럼 oligos의 집합 설계 되었습니다. 높은 중 합 효소를 사용 하 여, 닌 발기인 조각 증폭 되었다, (그림 1B), 절연 그리고에 transcriptional 퓨전을 생성 ...

Watch this Scientific Journal Video about Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean at JoVE.com

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

식물 발기인 분석: 식별 및 일반적인 콩에서 루트 결 절 특정 발기인의 특성

발기인 식 분석은 유전자 규정의 이해 및 대상 유전자의 spatiotemporal 식 개선에 중요 한. 여기 우리는 식별, 격리, 식물 발기인을 복제 하는 프로토콜을 제시. 또한, 우리는 일반적인 콩 털이 뿌리에 결 절 특정 발기인의 특성을 설명합니다.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

plant-promoter-analysis-identification-characterization-root-nodule

Research

JoVE Journal

Biology

11.7K 조회수.  Escuela Nacional de Estudios Superiores Unidad León- Universidad Nacional Autónoma de México (UNAM). 발기인 식 분석은 유전자 규정의 이해 및 대상 유전자의 spatiotemporal 식 개선에 중요 한. 여기 우리는 식별, 격리, 식물 발기인을 복제 하는 프로토콜을 제시. 또한, 우리는 일반적인 콩 털이 뿌리에 결 절 특정 발기인의 특성을 설명합니다.

비디오: Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

특정 엔도와 exoglycosidases를 사용하여 단백질 글리코 실화의 식별 및 특성

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

연구

생물학

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

형광 - 현미경 검사와 차세대 장면 : Organelle 무결성에 관련된 유전자의 식별을위한 유용한 도구

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

미생물 식별 및 특성화를위한 Pyrosequencing

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

식물 단백질 복합체의 번역 후 변형의 확인

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

플라스미드 벡터를 생성 깁슨 어셈블리를 사용하여 인간의 신장 요인-1α의 발기인의 규정에 따라 FLAG-태그 단백질을 표현

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

의 측면 루트 유도 성 시스템<em&gt; 애기 장대</em&gt;과 옥수수

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

방법은 쥐 Bcrp1의 대체 발기인 사용 및 전사 규정을 발견 할 수

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

헬리안투스 아누스의 대규모 열화상 스크리닝에 의한 새로운 식물 증발 규제 기관 식별

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

레이저 캡처 미세 절 RNA-시퀀싱 용 공간 및 측두 조직 -특정 유전자 발현 분석

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

가중 상관 관계 네트워크에 따라 다른 서식지에서 루트 미생물의 발산

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...