이 작품은 와게닝겐 대학 기금(WUF), 대학원생을 위한 중국 장학위원회 프로그램, NWO-VIDI 교부금 12378에 의해 부분적으로 지원됩니다.

1. 식물 재배 및 테스트 조건
<ol><li>18-22°C의 온도 범위 내에서 또는 자연 광 정권 하에서 또는 16 시간/ 8 시간 낮/밤 정권으로 제어 온실 또는 기후 챔버에서 식물을 성장하고 유지합니다. 식물을 보다 관리하기 위해 축실 가지를 제거하십시오.</li>
	<li>감자의 경우, 무라시게-스쿠그(MS) 매체(20g/L 자당, 비타민 5g/L/L MS 염, 8g/L agar, pH 5.8)를 함유한 멸균 플라스틱 항아리에 체외질을 유지하여 18°C의 기후챔버에서 16시간/8시간/1박/야간 으로 16시간/8시간 동안 온실로 이송하여 오염된 토양으로 이송한다.</li>
</ol>2. 농약침
<ol><li>식물 재료: 니코티아나 벤타미안의경우, 약 4-5주 된 종자 재배 식물을 사용하십시오. 감자의 경우 체외에서 4-5주 된 이식을 사용하십시오. 침투를 위해 젊고 건강하며 완전히 발달된 잎을 선택하십시오.</li>
	<li>
아그로바테리움 문화:<ol>
<li>YEB 배지를 미리준비합니다(표 1). 50ml 튜브를 1μl 아세토시링게오네(200mMM), 100 μl MES 버퍼(2-(N-morpholino)-에탄 설포닉산, 1M) 및 적절한 항생제로 보충한 10ml YEB 배지로 튜브를 채웁니다. 파이펫 20 μl 글리세롤 스톡은 원하는 균주의(표 2)(관심 유전자 함유)을 YEB로 한다. 동시에이 분석에서 모든 Agrobacterium 균주에 대한 문화를 시작합니다. 세포를 28°C에서 1-2일 동안 흔들어 서 200rpm에서 약 1.0의 OD600으로 배양한다.</li>
		<li>원심분리에 의해 10분 동안 3,000 x g에서 세포를 수확하십시오. 상체를 부어 신선하게 만든 MMA 배지(표 3)에서 펠릿을0.3의OD600으로 재연한다. 부드럽게 소용돌이 세포를 다시 중단합니다. 두 개의 세균성 균주의 동전 여과의 경우 배양을 1:1 비율로 혼합하십시오.</li>
		<li>침투하기 전에 1-6 시간 동안 실온에서 벤치에 문화를 둡니다. 그 동안, 식물에 침투 할 및 실험 날짜를 레이블.</li>
	</ol>
</li>
	<li>침투: 1ml 바늘없는 주사기를 사용하여 Agrobacterium 현탁액에 침투하십시오. 주사기의 현탁액이 있는 잎 패널을 주의 깊게 천천히 주입합니다(건강 및 안전상의 이유로 이 과정에서 눈 보호가 착용되어야 합니다). 각 변형에 대해 적어도 3 개의 식물에 침투합니다. 식물 당 3 개의 잎을 사용하여 삼중 으로 사용하십시오. 참고 : 일반적으로, Agrobacterium 서스펜션 의 1 ml는 N. 벤타미안의3 식물에 충분 할 수있다. 감자의 경우 Solanum 종의 잎이 침투하기가 더 어렵기 때문에 더 많은 현탁액이 필요합니다. 서스펜션의 필요한 볼륨은 솔라눔 종에 따라 달라집니다. 장갑을 교체하거나 침투 사이에 에탄올로 장갑을 살균하고 접종 다음날까지 식물에 물을 주지 않음으로써 교차 오염을 피하십시오.</li>
	<li>점수: 세포사멸 표현형이 명확할 때 침투 후 약 3일 동안 거시적 반응점수(도1). 세포사멸 표현형이 정량적인 경우 주어진기준(도 2)을사용한다. 간략하게, 세포 죽음의 거시적인 점수에 대한 비늘은 주로 침투 된 지역의 세포 사멸 비율에 달려 있습니다. 세포 사멸의 백분율은 0%(증상 없음)에서 100%(컨실런사)까지 의 척도로 묘사됩니다. 중간 반응은 클로로시스와 같은 약한 반응에서 세포 사멸의 증가 수준에 이르기까지 다양합니다. 원하는 경우 거시적 점수는 현대적인 사진 이미징 장비를 사용하여 정량적으로 평가될 수도 있습니다.</li>
</ol>3. PVX 농병 소독
<ol><li>식물 재료: N. 벤타미안의경우, 약 2-3 주 된 종자 재배 식물을 사용하십시오. 감자의 경우 체외에서약 2-3 주 된 이식을 사용하십시오. 대규모 테스트의 경우, 약간 오래 된 (4-5 주) 식물을 사용, 이 식물은 Agrobacterium 접종 반점의 높은 숫자를 수용 하기 위해 점점 더 큰 잎으로.</li>
	<li>
농균 배양:<ol>
<li>YEB 배지를 미리 준비하십시오. 10ml 튜브를 적절한 항생제로 보충한 3ml YEB로 채웁니다. 파이펫 20 μl 글리세롤 스톡은 원하는 균주의(표 2)(관심 유전자 함유)을 YEB로 한다. 동시에이 분석에서 모든 Agrobacterium 균주에 대한 문화를 시작합니다. 세포를 28°C에서 1-2일 동안 흔들어 서 200rpm에서 약 1.0의 OD600으로 배양한다.</li>
		<li>각 농균균균의 약 300 μl을 1-2일 동안 적절한 항생제로 보충하고 세포를 28°C로 배양하여 LB 고체 천기 중플레이트에 퍼뜨려 줍니다.</li>
	</ol>
</li>
	<li>감염: 주걱을 사용하여 접시에 있는 아그로박테리움 문화를 수집하십시오. 주걱에 Agrobacterium 문화에 나무 이쑤시개를 찍어 많은 양의 박테리아를 접종하기 위해 잎을 관통하십시오. 각 균주에 대해 적어도 3개의 식물을 접종합니다. 식물 당 3 개의 잎을 사용하여 삼중 으로 사용하십시오. 각 잎에서 각 변형에 대해 여러 예방 접종 부위를 만듭니다.</li>
	<li>점수: 접종 후 약 2주 동안 매크로 응답점수(그림 3). 처리량이 높은 화면의 경우 각 접종 지점에 대한 정성적 응답(예/아니요)을 기록합니다. 그런 다음 응답 사이트의 백분율을 계산하고 컨트롤과 비교합니다.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+transformation-competent+Arabidopsis+genomic+library+in+Agrobacterium.">A DNA transformation-competent Arabidopsis genomic library in Agrobacterium.</a> Bio-Technol. 9, 963-967 (1991).</li><li>Rietman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Putting the Phytophthora infestans genome sequence at work; multiple novel avirulence and potato resistance gene candidates revealed [PhD thesis]. , (2011).</li><li>Wroblewski, T., Tomczak, A., Michelmore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Agrobacterium-mediated+transient+assays+of+gene+expression+in+lettuce,+tomato+and+Arabidopsis.">Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis.</a> Plant Biotechnol. J. 3, 259-273 (2005).</li><li>van engelen, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pBINPLUS+-+an+improved+plant+transformation+vector+based+on+pBIN19.">pBINPLUS - an improved plant transformation vector based on pBIN19.</a> Transgenic Res. 4, 288-290 (1995).</li><li>Karimi, M., Inze, 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=GATEWAYTM+vectors+for+Agrobacterium-mediated+plant+transformation.">GATEWAYTM vectors for Agrobacterium-mediated plant transformation.</a> Trends Plant Sci. 7, 193-195 (2002).</li><li>Xiang, C. B., Han, P., Lutziger, I., Wang, K., Oliver, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mini+binary+vector+series+for+plant+transformation.">A mini binary vector series for plant transformation.</a> Plant Mol. Biol. 40, 711-717 (1999).</li><li>Curtis, M. D., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gateway+cloning+vector+set+for+high-throughput+functional+analysis+of+genes+in+planta.">A gateway cloning vector set for high-throughput functional analysis of genes in planta.</a> Plant Physiol. 133, 462-469 (2003).</li></ol>

저자는 경쟁적인 재정적 이익이 없다고 선언합니다.

농약과 농병과 같은 일시적인 연구는 현대 분자 식물 병리학 연구에 필수적인 효율적인 방법입니다. 몇 가지 제한에도 불구하고 이러한 방법은 플랜트에서 효율적이고 견고한 고처리량 기능 분석에 대한 수요를 충족시다.
농구침 시스템은 식물 종의 범위에서 널리 사용되는 기능 분석이다. 농약은 상호 작용하는 단백질의 동시 발현을 가진 몇몇 전간 유전자의 전달을 동일 세포로 전달하는 것을 용이하게 합니다. 이것은 일치하는 R 유전자를 표현하는 긴장으로 Avr 유전자를 표현하는 Agrobacterium 균주를 동전으로 하여 R-AVR 관계를 재구성하는 데 유리합니다. 또한 알려진 R-AVR 쌍의 경우 이러한 코인 여과를 긍정적 인 컨트롤로 사용할 수 있습니다. 일부 식물 유전자형에서는 변환 효율이 응답을 감지하기 위해 임계값 이하로 있을 수 있기 때문에 이러한 컨트롤을 포함하는 것이 중요합니다. 부정적인 대조를 포함하되, 예를 들어 유전자 삽입 없이 벡터를 함유하는 Agrobacterium 균주는 특정 식물 유전자형이 Agrobacterium에대한 비특이적 방어 반응을 제기하는지 여부를 결정하는 데 필수적이다. 이 기능은 감자 세균의 특정 주파수에서 발생하며 모든 솔라넘 종이 이 Agrobacterium기반 발현 시스템에 적합하지는 않습니다. 일반적으로, 농약 침투 분석은 N. 벤타미안과 대부분의 감자 유전자형에서 아주 잘 작동합니다. 이펙타로믹스 외에도, 공초점 현미경에 의한 식물 세포에서 트랜스게놈및 단백질 국소화에서 단백질의 생산과 같은 농구침 기술에 대한 다양한 잠재적 응용 이있다.
PVX 농병은 고감도 스크리닝 시스템이며 일반적으로 고처리량 스크리닝에 더 적합합니다. Agrobacterium은 지금 단지 로컬 존재 이기 때문에, 이 박테리아에 비특이적인 반응은 지금 아주 방해하지 않습니다, PVX 바이러스는 질균의 추가 퍼짐을 인수하기 때문에. 그러나, 식물은 PVX에 내성이 있거나, 극단적인 저항(ER) 반응을 탑재할 수 있으며, 이 경우 농아소독 방법이 적합하지 않다. PVX 농사감염 방법의 또 다른 제한은 관심 있는 유전자의 삽입 크기이다. 관찰된 표현형의 반응은 상처를 둘러싼 강렬한 흑괴사에서 접종 지점 근처의 희미한 괴사에 이르기까지 다양할 수 있다. N. 벤타미나와 솔라눔 종 모두에서 PVX 농소독은 농구침보다 더 민감하다는 것을 인식합니다.
다양한 테스트 된 식물 유전자형의 유전 적 배경이 몇 가지 제한이있을 수 있다는 점을 고려하여 (위 참조), 우리는 일반적으로 PVX 농가 감염 및 농생물 침투에 의해 유사한 결론을 얻을. 이러한 결과는 또한 단백질침투(29) 및 ELISA3과같은 다른 애사에서 얻은 것과 비교할 수 있다. 두 시스템의 장점과 한계를 고려하여 두 방법을 모두 사용하여 서로를 보완하거나 독립적인 결과를 확인하는 것이 좋습니다.

이펙토로믹스, 고처리량 기능유전체학 접근법은 최근 작물 식물에서내성(R)유전자를 식별하고병원균의유전자(Avr)유전자를 식별하는 강력한 도구로 부상하고 있다. R 유전자를 가진 더 많은 시간 소모적인 안정한 변환과는 대조적으로, 이펙소학 전략은 병원체 유전자 서열의 일시적인 분석에 근거를 두고 있습니다.
유전체학 시대 부터 식물 병원균의 게놈이 널리 탐구되었습니다. 예를 들어 가장 파괴적인 식물 병원균을 포함하는 oomycetes를 위해, 식물5-10과의상호 작용 도중 역할을 하는 유전자를 위해 서열의 큰 집합이 생성되고 분석되었습니다. 병원체 단백질의 한 클래스는 감염 (독성 요인)을 용이하게하거나 방어 반응 (비보렌스 인자)11-13을트리거하기 위해 숙주 세포 구조와 기능을 조작하는 이펙터를 나타낸다. R 유전자를 포함하는 식물 세포에서 Avr 유전자의 발현은 일반적으로 과민성 세포 사멸 반응 (HR)14,15의결과를 낳는다. R 및 Avr 유전자의 질문 발현에서 아그로박테리움 투메파시엔과같은 일시적인 발현 시스템을 사용하여 달성될 수 있다- 기반과도 변환(agroinfiltration)16. 이러한 일시적인 변환은 또한 바이러스 발현 시스템(agroinfection)17,18과함께 적용될 수 있다.
농구침의 경우, 가장 일반적으로 사용되는 제제는 디코 식물의 광범위한 숙주 범위 병원균인 A. tumefaciens입니다. A . tumefaciens는 종양 유도 (Ti) 플라스미드를 포함합니다. 티 플라스미드로부터 의 전DNA(T-DNA)는 박테리아의 독성 기계가 활성화된 후 식물 세포로 전이된다. 이는 상처입은 식물 세포에서 발동될 수 있으며, 약간 산성 환경에서 방출된 저분자-중량 페놀 화합물 및 단색차르에 의해19일. 독성 유전자는 주요 정맥에 의해 정의된 잎 패널로 Agrobacterium 현탁액의 침투 후에 활성화됩니다. 그런 다음 잎 패널내의 식물 세포가 T-DNA 영역에 포함된 트랜스진을 변형시키고 발현하게 됩니다.
농병은 세포를 심기 위하여 바이러스의 전좌를 중개하는 상처 접종한 Agrobacterium를기반으로 합니다. 바이러스는 그 때 Agrobacterium의부재에, 인접 한 식물 조직에 더 확산. 농가 감염의 경우 여러 식물 바이러스를 사용할 수 있습니다. RNA 바이러스는 감염된 식물에서 매우 높은 수준으로 증식할 수 있기 때문에 유전자 발현에 이상적인 벡터입니다. 식물 RNA 바이러스 중, 감자 바이러스 X (PVX)는 이펙소로믹스 스크린에 널리 사용된다. 삽입된 유전자에 대한 기능성 검사를 용이하게 하기 위해 콜리플라워 모자이크 바이러스(35S 프로모터)와 노팔린 신타제 터미네이터에 의해 측면에 있는 PVX 게놈을 포함하는 이진 벡터는 A.tumefaciens20의T-DNA로 복제되었다. T-DNA가 식물 세포로 옮겨진 후, T-DNA에 포함된 PVX 게놈은 35S 프로모터로부터 전사된다. 그런 다음 바이러스 입자가 감염된 식물에서 체계적으로 퍼지고 삽입된 유전자의 발현을 초래합니다. 이 방법은 아그로박테리움과 PVX를 기반으로 PVX 농사독이라고 합니다.
여기서 우리는 농아침투와 PVX 농소소 소독 소사 모두에 대한 예를 보여줍니다. 호스트 식물로서 우리는 감자 세균(솔라눔 섹션 Petota)을사용하며, 이펙토로믹스 접근법이 개척되어3,4의성공을 입증했습니다. 우리는 또한14,21,22의 솔라나체 식물의 모델 공장으로 유명한 니코티아나 벤타미안을사용합니다.

<table border="0" cellpadding="0" cellspacing="0" width="955">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Beef extract</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">B4888</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Bacteriological peptone</td>
			<td style="width:267px;">Oxoid</td>
			<td style="width:219px;">LP0037</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Yeast extract</td>
			<td style="width:267px;">Oxoid</td>
			<td style="width:219px;">LP0021</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MgSO4</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">208094</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MS salts (without vitamins)</td>
			<td style="width:267px;">Duchefa Biochemie</td>
			<td style="width:219px;">M0221</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">MES</td>
			<td style="width:267px;">Duchefa Biochemie</td>
			<td style="width:219px;">M1503</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">LB broth powder</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">L3022</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Acetosyringone</td>
			<td style="width:267px;">Sigma-Aldrich</td>
			<td style="width:219px;">D134406</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Syringe (1 ml)</td>
			<td style="width:267px;">BD Plastipak</td>
			<td style="width:219px;">300013</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Incubator</td>
			<td style="width:267px;">Infors HT</td>
			<td style="width:219px;">Multitron II</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Centrifuge</td>
			<td style="width:267px;">Heraeus</td>
			<td style="width:219px;">Multifuge 3S-R</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:252px;">Spectrophotometer</td>
			<td style="width:267px;">Eppendorf</td>
			<td style="width:219px;">Biophotometer 6131</td>
			<td style="width:219px;"></td>
		</tr>
	</tbody>
</table>

agroinfiltration and pvx agroinfection in potato and nicotiana benthamiana

농약침및 PVX 농병소는 식물에 있는 후보 유전자의 기능적 분석을 위한 2개의 능률적인 일시적인 발현 분석이다. 농구침을 위해 가장 일반적으로 사용되는 에이전트는 많은 디코트 식물 종의 병원체인 아그로박테리움 tumefaciens입니다. 이것은 농약 침전이 많은 식물 종에 적용될 수 있다는 것을 의미합니다. 여기서, 우리는 감자(솔라눔 tuberosum),관련 야생 결핵 베어링 솔라눔 종(솔라눔 섹션 Petota)과모델 식물 니코티아나 벤타미안에이러한 방법을 적용 할 때 우리의 프로토콜과 예상 결과를 제시한다. 저항성(R)또는 avirulence(Avr) 유전자와 같은단일 유전자의 기능적 분석 외에도, 농약침 분석은 단순히 R 및 Avr 전진을 동일한 세포로 전달함으로써 특정 숙주 병원체 상호 작용과 관련된 R-AVR 상호 작용을 재구성하는 데 매우 적합합니다. 그러나, 몇몇 식물 유전형은 우리가 몇몇 감자 유전자형에 대해 예를 들었던 바와 같이, Agrobacterium에비특이적인 방어 반응을 올릴 수 있습니다. 농약침과 비교하여 PVX 농약으로 AVR 활동을 감지하는 것은 기능성 스크린에서 더 민감하고, 더 높은 처리량이며, Agrobacterium에대한 비특이적 방어 응답에 덜 민감하다. 그러나 PVX에 대한 비특이적 방어가 발생할 수 있으며 바이러스유발 극단적인 저항으로 인해 응답을 놓칠 위험이 있습니다. 이러한 한계에도 불구하고, 우리의 경험에서, 농침과 PVX 농병은 서로의 결과를 확인하기 위해 동시에 사용할 수있는 적합하고 보완적인 분석입니다.

도 1은 감자 및 N. 벤타미안에서 7가지 이펙터(E1-E7)를 가진 농약침투의 대표적인 실험을 나타낸다. 세포 사멸은 침투 후 약 3 일 후에 침투된 잎 패널에 나타납니다. 세포 사멸 표현형의 범위는 대조군과 비교될 필요가 있다. PBINplus-R3a 및 pK7WG2-AVR3a를 함유한 아그로박테리움 균주 AGL1(pVirG)23의 혼합물은 양성 대조군으로사용되었고, AGL1(pVirG)은 음의 대조군으로 사용되었다. 도 1A 및 1B는 감자 유전자형 MaR8(Mastenbroek R8)26 및 N. 벤타미안나에서농약침의 좋은 예를 보여주며, 이는 농가 침투에 매우 적합합니다. 양극 대조군으로 혼입된 잎 패널에 컨실루션 세포사멸이 있으며, 음의 대조군 또는 pBINplus-R3a로 세포 사멸 반응이 발생하지 않습니다. MaR8에서, 2개의 이펙터 AVR3a(E2) 및 AVR4(E3)는 세포 사멸을 유도하고, 다른 이펙터(E1 및 E4-E7)는 그렇지 않습니다. 도 1C 및 1D는 야생 감자 솔라눔 베르타울리 483-1및 솔라눔 rechei 210-5에서 농아침투의 예를 보여 주며, 이는 농아침투 기술에 적합하지 않다. S. berthaultii 483-1에서, 잎 조직은 모든 시험된 이펙터뿐만 아니라 부정적인 통제에 비특이적 괴사를 보여줍니다. S. rechei 210-5에서, 침투된 잎 패널은 긍정적인 통제에 매우 약한 세포 죽음 반응을 보여줍니다.
도 2는 농약침에 대한 반응을 정량화하는 데 사용할 수 있는 다양한 채점 스케일을 나타낸다. 세포 사멸의 백분율은 0-100%의 척도로 묘사됩니다. 관찰된 표현형은 거시적으로 눈에 보이지 않는 증상(0%)부터 클로로시스를 표시하고 세포 사멸 수준을 증가시키는 중간 반응의 범위를 통해 컨서블세포 사멸(100%)까지 다양합니다.
도 3은 감자에서 PVX 농소염 후 대표적인 실험을 나타낸다. 일반적으로, 확장 세포 죽음은 치아 선택 접종 후에 대략 2 주 사이트에서 찾아낼 수 있습니다. 두 그림 패널에 도시된 바와 같이, 세포 사멸확대는 PGR106-CRN2(이펙터 크링클러 Crn2를 이용한 양성 대조군)로 접종된 치아 선택 부위에 존재하며, 이는 P. infestans27로부터유전자를 유도하는 일반적인 세포 사이다. 상처에 대한 사소한 반응 외에도 pGR106-empty (부정적인 대조군)로 접종 된 치아 선택 부위에서 확장 세포 사망이 지적되지 않습니다. 도 3A에서감자 유전자형 MaR3(Mastenbroek R3)를 나타내는 2개의 pGR106-이펙터(E1 및 E2)는 세포 사멸을 유도하지 않았다. 도 3B에서,야생 솔라눔 huancabambense 354-1에서 이펙터 스크리닝의 긍정적인 결과가 제시된다; 세포사멸 반응은 2개의 pGR106-이펙터(E1-E3, 유도를나타내는)(28)로관찰되었다.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50971/50971fig1highres.jpg" src="/files/ftp_upload/50971/50971fig1.jpg"> 그림 1. 감자와 N. 벤타미안의 농약 침투의 예. 식물 포함(A)감자 유전자형 MaR8 (마스텐 브룩 R8),(B) N. 벤타미안,(C) 솔라눔 베르타울리 483-1 및(D) 솔라눔 rechei 210-5에 침투했다 PBINplus-R3a 및 pK7WG2-AVR3a(양성 제어), AGL1(pvirG) (음의 제어), pBINplus-R3a를 함유한 아그로박테리움 균주 AGL1(pVirG) 23의 혼합물, 및 7개의 이펙터(E1-E7). <a href="https://www.jove.com/files/ftp_upload/50971/50971fig1highres.jpg" target="_blank">더 큰 이미지를 보려면 여기를 클릭하십시오.</a>
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50971/50971fig2highres.jpg" src="/files/ftp_upload/50971/50971fig2.jpg"> 그림 2. 농약 침전 반응의 정량화. 사진은 세포 사멸을 위한 대표적인 채점 척도를 보여줍니다, 0%에서 100% (confluent 세포 죽음). 중간 반응은 클로로시스와 같은 약한 반응에서 세포 사멸의 증가 수준에 이르기까지 다양합니다. <a href="https://www.jove.com/files/ftp_upload/50971/50971fig2highres.jpg" target="_blank">더 큰 이미지를 보려면 여기를 클릭하십시오.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971fig3highres.jpg" src="/files/ftp_upload/50971/50971fig3.jpg"> 그림 3. 감자에서 PVX 농가소의 예. 감자 유전자형 MaR3 (마스텐브룩 R3)(A)및 솔라눔 huancabambense 354-1(B)치아 선택 pGR106-CRN2 (양성 제어), pGR106-빈 (네거티브 컨트롤) 및 pGR106-E1-2 (효과) 또는 pGR106-E1-2 (효과) 또는 pGR106-E1-2 (효과) 또는 pGR106-E1-2(효과제), 또는 pGR106-E1-2(효과) 또는 pGR106-EGR1-EGR106-EGR1-EGR106-EGR1-EGR106 <a href="https://www.jove.com/files/ftp_upload/50971/50971fig3highres.jpg" target="_blank">더 큰 이미지를 보려면 여기를 클릭하십시오.</a>
표 1. YEB 매체
<table border="1"><tbody>
<tr>
<td>1 L</td>
			<td>증류H2O</td>
		</tr>
<tr>
<td>5 g</td>
			<td>자당</td>
		</tr>
<tr>
<td>5 g</td>
			<td>쇠고기 추출물</td>
		</tr>
<tr>
<td>5 g</td>
			<td>세균성 펩톤</td>
		</tr>
<tr>
<td>1 g</td>
			<td>효모 추출물</td>
		</tr>
<tr>
<td>2 ml</td>
			<td>MgSO4 (1 M)</td>
		</tr>
</tbody></table>표 2. 농약 과 PVX 농업 소독에 사용되는 벡터 및 균주. 여러 이진 벡터를 사용할 수 있습니다. 아래 목록에 있는 벡터는 후보 유전자의 높은 발현을 허용하고 우리의 손에 잘 일했습니다. 우리는 N. 벤타미안에서 Agrobacterium 균주 GV3101 (pMP90)29를 사용하는 것을 선호하고 조퍼 플라스미드 pVirG (pBBR1MCS-5.virGN54D)23 감자31에더 적합한 헬머 플라스미드 pVirG를 포함하는 AGL130을 찾을 수 있습니다. 추가 균주는 N. 벤타미안을 포함하여 각종 모형 식물에 그밖 단에서 분석되었습니다 그러나 감자(32)에서는아닙니다.
<img alt="Table 2" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971table2highres.jpg" src="/files/ftp_upload/50971/50971table2.jpg">
GW: 게이트웨이 버전36
표 3. MMA 매체. <img alt="Table 3" fo:content-width="5in" fo:src="/files/ftp_upload/50971/50971table3highres.jpg" src="/files/ftp_upload/50971/50971table3.jpg">
pH를 5.6으로 조정합니다.

Watch this Scientific Journal Video about Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana at JoVE.com

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

농약침과 PVX 농병은 식물에 있는 유전자의 일시적인 자궁 발현을 위한 일상적인 기능적인 분석입니다. 이 방법은 효과적인 전략 (급속한 저항 및 비열유전자 발견)에 있는 능률적인 연구및 분자 식물 병리학에 있는 현대 연구에 결정적입니다. 그들은 공장에서 강력한 고처리량 기능 분석에 대한 수요를 충족합니다.

감자와 니코티아나 벤타미안의 농아침투 및 PVX 농병감염

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

agroinfiltration-pvx-agroinfection-potato-nicotiana

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Biology

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

20.2K 조회수.  Wageningen University. 농약침과 PVX 농병은 식물에 있는 유전자의 일시적인 자궁 발현을 위한 일상적인 기능적인 분석입니다. 이 방법은 효과적인 전략 (급속한 저항 및 비열유전자 발견)에 있는 능률적인 연구및 분자 식물 병리학에 있는 현대 연구에 결정적입니다. 그들은 공장에서 강력한 고처리량 기능 분석에 대한 수요를 충족합니다.

비디오: Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

바이러스 유도 진 입을 (VIGS)에 Nicotiana benthamiana 토마토

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

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생물학

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Murine 조혈 줄기 세포와 리니지 - 헌신 Progenitors의 Phenotypic 분석 및 절연

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

재조합 단백질의 높은 수준의 과도 발현 식물의 효율적인 Agroinfiltration

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

최적화 및 활용<em&gt; 아그로 박테 리움</em에서&gt; 매개 과도 단백질 생산<em&gt;는 담배</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

총회와 &#39;전단 반지&#39;의 응용 프로그램 : 궤도, 단방향 및 정기 유체 흐름 및 전단 응력에 대한 소설 내피 모델

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Luciferase 보완성 이미징 분석 결과 Nicotiana benthamiana 잎 뚜렷이 결정 단백질 단백질 상호 작용 역동성에 대 한

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant growth-promoting rhizobacteria (PGPR) as biofertilizers or biocontrol agents has become an effective alternative to the use of conventional fertilizers and can increase crop productivity at low cost. Plant-microbe interactions depend upon host plant-secreted signals and a reaction hereon by their associated bacteria. However, the molecular mechanisms of how beneficial bacteria respond to their associated plant-derived signals are not fully understood. Assessing the transcriptomic response of bacteria to root exudates is a powerful approach to determine the bacterial gene expression and regulation under rhizospheric conditions. Such knowledge is necessary to understand the underlying mechanisms involved in plant-microbe interactions. This paper describes a detailed protocol to study the transcriptomic response of B. mycoides EC18, a strain isolated from the potato endosphere, to potato root exudates. With the help of recent high-throughput sequencing technology, this protocol can be performed in several weeks and produce massive datasets. First, we collect the root exudates under sterile conditions, after which they are added to B. mycoides cultures. The RNA from these cultures is isolated using a phenol/chloroform method combined with a commercial kit and subjected to quality control by an automated electrophoresis instrument. After sequencing, data analysis is performed with the web-based T-REx pipeline and a group of differentially expressed genes is identified. This method is a useful tool to facilitate new discoveries on the bacterial genes involved in plant-microbe interactions.

Plant-Microbe Interaction: Transcriptional Response of Bacillus Mycoides to Potato Root Exudates

The goal of the protocol presented here is to study the transcriptomic response of endosphere-isolated Bacillus mycoides to potato root exudates. This method facilitates the identification of important bacterial genes involved in plant-microbe interactions and is in principle applicable to other endophytes and plants, with minor adjustments.

감자 루트 Exudates 균 Mycoides 의 Transcriptional 응답을 식물 세균 상호 작용:

Beneficial plant-associated bacteria play an important role in promoting growth and preventing disease in plants. The application of plant ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2단계 PCR 및 차세대 16S rRNA 유전자 시퀀싱을 사용한 미생물 분석

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

니코티아나 벤타미안나에서 과도하게 표현된 IgG 융합 단백질의 생산

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...

감자와 니코티아나 벤타미안의 농아침투 및 PVX 농병감염

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