CN113265385B - 构树抗菌蛋白BpChiI及其重组表达载体和提高植物对黄萎病抗性中的应用 - Google Patents
构树抗菌蛋白BpChiI及其重组表达载体和提高植物对黄萎病抗性中的应用 Download PDFInfo
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- CN113265385B CN113265385B CN202110574197.XA CN202110574197A CN113265385B CN 113265385 B CN113265385 B CN 113265385B CN 202110574197 A CN202110574197 A CN 202110574197A CN 113265385 B CN113265385 B CN 113265385B
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- verticillium wilt
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- broussonetia papyrifera
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Abstract
本发明公开了构树抗菌蛋白BpChiI及其重组表达载体和提高植物对黄萎病抗性中的应用，构树抗菌蛋白BpChiI具有SEQ ID NO.18所示的氨基酸序列，核苷酸序列如SEQ ID NO.17所示，BpChiI对植物病原真菌具抑菌作用；利用根癌农杆菌介导法将其整合到植物基因组中，对黄萎病的抗性显著提高，具有巨大的应用前景。
Description
技术领域
本发明涉及植物基因工程领域，具体涉及构树抗菌蛋白BpChiI，含涉及含有编码构树抗菌蛋白BpChiI基因的重组表达载体，以及构树抗菌蛋白BpChiI在提高植物对黄萎病抗性中的应用。
背景技术
植物病害是长期危害农业生产的自然灾害之一，它不仅引起农作物减产，而且严重威胁到农产品的质量、食品安全和国际贸易，严重时还会引起食物短缺和一系列社会问题(Punja,2004；Agrios G et al.,2005；Gisi U.et al.,2009)。植物病害多种多样，其中真菌病害最广泛，危害最为严重。
黄萎病是一种世界性的病害，从温带、亚热带到热带地区均有黄萎病发生的报道(Pegg GF,2002)。其致病菌是一种土传维管束病原菌，变异频率快，生理小种多，在土壤中可以存活20年之久，能感染200多种植物品种，除单子叶植物以外的所有植物，包括各种蔬菜、果树、农作物、林木、花草等等都是黄萎病菌的寄主，每年全球因黄萎病引起的作物产量损失就达数十亿美元，其中，马铃薯感染黄萎病菌其损失可以达到50％以上，生菜非常容易达到100％，棉花在黄萎病发病严重的年份也容易造成颗粒无收，在所有维管束病害中，黄萎病菌引起的病害损失是最严重的(Pegg GF,2002；Steven J.Klosterman等,2009；AgriosG，2005)。目前，克隆获得的抗黄萎病基因只有来自番茄的Ve1，该基因在生菜中也存在，但是几年之后抗性也丧失，更为重要的是，绝大多数作物缺乏抗黄萎病的抗原，并且难以获得具有抗性的抗原(Steven J.Klosterman等,2009)。
实践证明，选育和推广使用抗病品种是最经济有效的防治措施，面对病原菌的快速变异和生理小种的特异性，创制具有广谱抗性的材料才是解决问题的根本。基因工程可以克服许多传统育种的不足，可使用的基因更多，来源更广。此外，基因工程还可以针对更大范围的病原菌获得更广谱的抗病性，对土壤有益微生物的影响也最小(Owen Wally等，2010)。截止目前已有较多利用转基因手段提高作物抗病性的报道，比如，在胡萝卜中超量表达来自木霉的几丁酶基因CHIT36提高了转基因植株对真菌病害的抗性(Baranski R等，2008)。在番茄和水稻中分别超量表达不同来源的防御素基因RsAFP2和DmAMP提高了它们的抗病性(Jha S等，2009)，在水稻中表达峰毒素基因提高了水稻对白叶枯病的抗性(WeiShi，2016)等等。但是获得的转基因材料仍然不能满足生产需求，尚无成功应用于生产的转基因抗病材料。基因不丰富，抗性不持久是一个重要原因，因此，进一步挖掘不同来源的基因具有重要意义。
植物抗病基因工程中，利用抗菌蛋白基因提高植物的抗病性一直受到人们的关注，在植物中表达不同来源的抗菌蛋白能显著提高其对病原菌的抗性(Shukurov,R.R etal.,2010；Kovács,G et al.,2013；Kaur,J et al.,2017)。抗菌蛋白不仅来源广泛，能直接作用于病原菌，而且作用于病原菌产生的降解产物也可以进一步诱导植物的防御反应，从而提高植物的抗病性。构树属于桑科构属，适应性强，分布广泛，抗逆性强，其树叶、乳液、果实、树皮等都有一定的药用价值(杨小建等，2007)。构树乳汁有治疗皮肤病的先例，但是未对起作用的组分进行分离。因此分离获得构树乳汁抗菌蛋白，阐明其抗病效果，克隆相关基因可以为植物抗病基因工程提供目的基因，对提高植物黄萎病抗性具有重要意义。
发明内容
有鉴于此，本发明的目的之一在于提供构树抗菌蛋白BpChiI；本发明的目的之二在于提供含有编码所述构树抗菌蛋白BpChiI基因的重组表达载体；本发明的目的之三在于提供所述构树抗菌蛋白BpChiI或所述重组表达载体在提高植物对黄萎病抗性中的应用；本发明的目的之四在于提供提高植物对黄萎病抗性的方法。
为达到上述目的，本发明提供如下技术方案：
1、构树抗菌蛋白BpChiI，所述构树抗菌蛋白BpChiI的氨基酸序列如SEQ ID NO.18所示。
优选的，编码所述构树抗菌蛋白BpChiI的核苷酸序列如SEQ ID NO.17所示，或SEQID NO.17所示核苷酸经过一个或几个碱基的取代且编码相同氨基酸的核苷酸序列。
2、含有编码所述构树抗菌蛋白BpChiI基因的重组表达载体。
优选的，所述重组表达载体由SEQ ID NO.17所示序列连入pLGN-35S-Nos质粒的SmaI和KpnI酶切位点处。
3、所述构树抗菌蛋白BpChiI或所述重组表达载体在提高植物对黄萎病抗性中的应用。
优选的，所述植物为拟南芥、烟草或棉花。
4、提高植物对黄萎病抗性的方法，将编码所述构树抗菌蛋白BpChiI的基因在植物中过表达，获得对黄萎病具有抗性的植物。
优选的，所述在植物中过表达的方法将编码构树抗菌蛋白BpChiI的基因构建成重组表达载体，然后通过农杆菌介导获得转基因植物。
优选的，所述重组表达载体由SEQ ID NO.17所示序列连入pLGN-35S-Nos质粒的SmaI和KpnI酶切位点处而得。
优选的，所述植物为拟南芥、烟草或棉花。
本发明的有益效果在于：本发明通过从构树乳汁中分离纯化抗真菌蛋白，然后进行肽指纹图谱分析，再根据肽段序列设计简并引物，利用RACE和YADE等分子生物学手段克隆获得目的基因BpChiI，然后构建基因的植物组成型表达载体，然后再利用基因工程方法，将BpChiI基因整合到拟南芥、烟草和棉花中，获得了正常转录表达的转基因拟南芥、烟草和棉花株系。与非转基因对照相比，BpChiI转基因拟南芥的病情指数可以由对照的47.96降至20.64；转基因烟草的病情指数可以由对照的73.33降至12.50；瞬时表达BpChiI棉花的病情指数可以由对照的73.17降至40.86。表明，BpChiI能显著提高植物对黄萎病的抗性，该发明对于促进BpChiI基因在植物抗病基因工程中的应用具有重要意义。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为BpChiI蛋白的纯化(A：过DEAE-Sepharose柱收集的蛋白液SDS-PAGE分析，其中的2为穿柱液；B：CM-Sepharose柱分离后收集样品的SDS-PAGE分析；C：分子筛分离收集的样品SDS-PAGE分析)。
图2为BpChiI抗真菌活性分析(平板内利用滤纸扩散法检测BpChiI的抗真菌活性；1：20mM磷酸盐缓冲液,pH6.2；2：60％丙酮沉淀构树乳汁的蛋白粗提液，总蛋白浓度为374.3μg/mL；3：54μg/mL纯化的BpChiI蛋白液；4：20μg/mL BSA对照)。
图3为BpChiI抑制油菜黑斑病菌孢子的萌发(A：不同浓度的BpChiI与油菜黑斑病菌孢子共培养不同时间的萌发率。数值为三次重复试验的平均值±SD。B：30μg/mL BpChiI与油菜黑斑病菌于1/10PDA培养基上共培养6h，8h和10h，孢子萌发情况)。
图4为超量表达BpChiI的pLGN-35S-BpChiI植物表达载体图谱。
图5为超量表达BpChiI基因提高拟南芥对黄萎病的抗性(A：不同转基因拟南芥株系中BpChiI的表达水平，数值为三个技术重复的平均值±SD；B：转基因和对照拟南芥接种黄萎病菌14天，不同病级的百分率，数值为三次重复试验的平均值；C：接种黄萎病菌14天，转基因和对照拟南芥的病情指数，数值为三次重复试验的平均值±SD；D：接种黄萎病菌14天，转基因和对照拟南芥的叶柄中黄萎病菌的相对含量，数值为三个生物学重复试验的平均值±SD；E：接种黄萎病菌14天，转基因和对照拟南芥的病症；Null：转基因拟南芥株系分离的非转基因植株作对照，BpChiI-2，BpChiI-5，BpChiI-6和BpChiI-7：独立的转基因株系；**：差异极显著(p<0.01)。
图6为超量表达BpChiI基因提高烟草对黄萎病的抗性(A：不同转基因烟草株系中BpChiI的表达水平，数值为三个技术重复的平均值±SD；B：转基因和对照烟草接种黄萎病菌14天，不同病级的百分率；数值为三次重复试验的平均值；C：接种黄萎病菌14天的，转基因和对照烟草的病情指数，数值为三次重复试验的平均值±SD；D：接种黄萎病菌14天，转基因和对照烟草的病症；Null：转基因烟草株系分离的非转基因植株作对照，BpChiI-2，BpChiI-5，BpChiI-9，BpChiI-13，BpChiI-14和BpChiI-22：独立的转基因株系。**：差异极显著(p<0.01)。
图7为瞬时表达BpChiI基因提高棉花对黄萎病的抗性(A：瞬时表达BpChiI和对照棉花子叶接种黄萎病菌5天，不同病级的百分率。数值为三次重复试验的平均值；B：接种黄萎病菌5天，瞬时表达BpChiI和对照棉花子叶的病情指数，数值为三次重复试验的平均值±SD；C，接种黄萎病菌5天，瞬时表达BpChiI和对照棉花子叶的病症)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1、构树抗菌蛋白BpChiI的纯化
(1)粗提纯
秋季于野外搜集构树乳汁，纱布过滤去除杂质，然后13000rpm离心再去除乳胶，上清液利用不同饱和度的丙酮进行梯度沉淀，获得40％、60％、70％和80％丙酮沉淀的蛋白。pH6.2的20mM PBS缓冲液溶解沉淀。然后以未沉淀的乳汁上清液为对照进行抗菌活性检测，结果显示60％，70％和80％丙酮沉淀的蛋白都具有抗菌活性，60％和70％沉淀的蛋白抗菌活性均较高。因此，进一步的以70％丙酮沉淀的蛋白为材料进行抗真菌蛋白的分离纯化。
接着将60％和70％丙酮沉淀的蛋白液再进一步用硫酸铵梯度沉淀并再次检测蛋白活性。结果显示，70％硫酸铵沉淀获得的蛋白保持其原有的抑菌活性。
(2)DEAE-Sepharose阴离子交换层析分离
70％硫酸铵沉淀的蛋白用pH6.2的20mM PBS缓冲液溶解，之后用脱盐柱脱盐，然后用DEAE-Sepharose阴离子交换层析柱进行分离，收集穿柱液和用含1M NaCl的20mM的磷酸缓冲液(pH6.2)进行直线梯度洗脱的洗脱液，整个过程的流速为5mL/min，每管收集3mL。收集液真空冷冻干燥成粉末，再用pH6.2的20mM PBS缓冲液溶解。蛋白液用脱盐柱脱盐后再检测抗菌活性并进行SDS-PAGE(聚丙烯酰胺凝胶)电泳检测，结果如图1中A所示。结果显示，穿柱液具有较强的抗菌活性。
(3)CM-Sepharose阳离子交换柱分离
紧接着用CM-Sepharose阳离子交换柱分离第二步中收集的穿柱液，同样的收集穿柱液和用含1M NaCl的20mM的磷酸缓冲液(pH6.2)进行直线梯度洗脱的洗脱液，仍以流速为5mL/min进行洗脱，每管收集3mL。收集的蛋白液真空冷冻干燥成粉末状，继续用pH6.2的20mM PBS缓冲液溶解。蛋白液用脱盐柱脱盐后再检测抗菌活性并进行SDS-PAGE电泳检测，结果如图1中B所示。结果显示，洗脱的第一个峰收集的蛋白具有抗菌活性。
(4)分子筛分离纯化
因为电泳检测显示上一步纯化的蛋白仍然存在多条蛋白带(图1中B)。为了进一步纯化抗菌蛋白，将上一步收集到的具抗菌活性的部分进一步用HiloadTM 16/600，SuperdexTM 75pg分子筛进行分离。经20mM磷酸缓冲液(pH6.2)充分平衡后上样，然后用含1M NaCl的20mM磷酸缓冲液(pH6.2)进行直线洗脱，流速为1mL/min，每管收集3mL。随着洗脱的进行，蛋白分子由大到小依次被分离开来，根据色谱峰收集对应的洗脱液，浓缩后检测蛋白纯化液的抑菌活性。此步分离出现了一个明显的收集峰，电泳检测该收集峰获得的蛋白已经比较纯，除了一条明显的蛋白带以外，其余蛋白带已不明显(图1中C)。表明获得的蛋白已经较纯。
将上述获得的较纯的蛋白进一步进行脱盐处理，并进行浓缩，收集浓缩管中浓缩后的蛋白液，-20℃保存备用。
实施例2、纯化的抗菌蛋白抗真菌活性检测
1、构树乳汁分离的抗菌蛋白能有效抑制多种植物病原真菌的生长
首先，利用滤纸扩散平板抑菌法检测构树来源的蛋白抗真菌活性。将纯化的蛋白浓度调整到54μg/mL，并以60％丙醇沉淀构树乳汁的蛋白粗提液、20mM磷酸盐缓冲液(pH6.2)和20μg/mL BSA(牛血清白蛋白)为对照，分别滴加10μL不同的蛋白液到放置到真菌边缘的滤纸圆盘上，待蛋白液扩散开后再滴加10μL，然后置26℃暗培养2d，观察抑菌结果。结果显示，利用构树乳汁纯化获得的抗菌蛋白能有效抑制油菜黑斑病菌(Alternariabrassicae)、烟草赤星病菌(Alternaria Alternate)、番茄早疫病菌(Alternariasolani)，黑白轮枝菌(Verticillium dahliae)，疫酶属的土豆疫霉菌(Phytophthorainfestans)和尖孢镰刀菌(Fusarium oxysporum)的生长(图2)。
2、构树乳汁分离的抗菌蛋白能有效抑制油菜黑斑病菌孢子的萌发
为了进一步明确纯化的蛋白对植物病原真菌生长的影响，以油菜黑斑病菌孢孢子为研究对象，利用不同浓度的蛋白液与油菜黑斑病菌孢子进行保湿共培养。不同时间节点分别统计孢子的萌发率。结果显示，7.5μg/mL的蛋白液就能有效抑制油菜黑斑菌孢子的萌发，其半致死浓度为15.0μg/mL。浓度越高抑菌效果越明显(图3，A)。孢子生长状态也显示，30μg/mL蛋白液与黑斑病菌共培养10h仅个别孢子萌发(图3，B)。
实施例3、构树抗菌蛋白基因的克隆
1、构树RNA的提取及cDNA的合成
取构树嫩叶，按照说明书的步骤，利用Aidlab公司的EASYspin Plant RNA Kit试剂盒提取构树的RNA。利用宝生物的反转录试剂盒合成一链cDNA，作为构树抗菌蛋白基因扩增的模板。
具体操作流程如下：取新鲜的构树叶片用液氮速冻并研磨成粉，取约100mg粉末盛装入无核酸酶的1.5mL离心管，立即加入RNA裂解液500μL，用移液枪反复吹打直到裂解物中无明显块状组织，然后加入300μL稀释液，颠倒离心管3-4次混匀，室温放置3-5min。12000rpm，离心5min后取上清液500μL于一新的1.5mL无核酸酶的离心管中，加入250μL无水乙醇，立即用移液器吹打混匀，然后将混和液转移入RNA吸附柱，10000rpm，离心1min，弃滤液，吸附柱内加入600μL漂洗液，重复漂洗2次后，将吸附柱转移到洗脱管上，在吸附柱内加入100μL无RNA酶和无DNA酶的水，放置约3min后，10000rpm，离心1min，收集洗脱的RNA溶液并保存于-80℃。
(1)利用简并引物扩增抗菌蛋白基因模板cDNA的合成
取7μL上述提取的RNA溶液中加入1μL无RNA酶的DNA酶，2μL无RNA酶的DNA酶缓冲液，混匀后PCR仪内42℃反应2min，然后加入4μL反转录酶缓冲液，1μL反转录酶，1μL反转录引物混和物，4μL无RNA酶无DNA酶的双蒸水，混匀后37℃反应15min合成cDNA，85℃反应5s，终止反应。合成的cDNA保存于-20℃。
(2)5’-RACE模板cDNA的合成
以构树RNA作为模板，加入人工合成引物5'-接头(5’-aagcagtggtatcaacgcagagtacgcggg-3’，SEQ ID NO.1)，按照SMARTer RACE 5’试剂盒说明书，利用SMART MMLVReverse Transcriptase进行反转录，获得5’-RACE cDNA。
(3)3’-RACE模板cDNA的合成
以构树RNA作为模板，加入人工合成引物3'-接头(5’-ccagtgagcagagtgacgaggactcgagctcaagctttttttttttttttt-3’，SEQ ID No.2)，按照SMARTer RACE试剂盒说明书，利用SMART MMLV Reverse Transcriptase进行反转录，获得3’-RACE cDNA。
2、构树抗菌蛋白基因的克隆
为了克隆获得构树乳汁内的抗菌蛋白基因，纯化的蛋白进行SDS-PAGE电泳，考马斯亮蓝染色后，切取目的条带送南京钟鼎生物技术有限公司进行肽指纹图谱检测。结果获得了与桑树几丁质酶基因同源的两个肽段，分别为GPIQLTWNYNYGQCGR和RYCDIFRI，根据肽段设计简并引物(5’-acn tgg aay tay aay tay gg-3’，SEQ ID No.3)和(5’-atn cgr aadatr tcr car ta-3’，SEQ ID No.4)，然后以构树cDNA为模板，PCR扩增获得抗菌蛋白基因326bp的部分序列。在此基础上，设计5’和3’端接头引物(5’-aag cag tgg tat caa cgcaga gta cgc ggg-3’，SEQ ID No.5)和(5’-cca gtg agc aga gtg acg agg act cga gctcaa gct ttt ttt ttt ttt ttt-3’，SEQ ID No.6)，5’和3’端RACE引物(5’-cta ata cgactc act ata ggg caa gca gtg gta tca acg cag agt-3’，SEQ ID No.7)和(5’-cta atacga ctc act ata ggg ccc agt gag cag agt gac gag gac-3’，SEQ ID No.8)，以及第一轮RACE的5’端下游特异引物(5’-att gag acc ttg gcc gca ttc gat g-3’，SEQ IDNo.9)，第一轮RACE的3’上游特异引物(5’-gct gtt att tcg ttc gag aca gcg c-3’，SEQID No.10)，第二轮RACE的5’端下游特异引物(5’-tcg atg cca ccg ttg atg at-3’，SEQID No.11)，第二轮RACE的3’上游特异引物(5’-cag cgc ttt ggt tct gga tg-3’，SEQ IDNo.12)。以上述这些引物为引物，构树cDNA为模板，将抗菌蛋白基因序列在326bp的基础上，分别向其5’和3’端延伸，根据测序序列进行拼接，分析拼接序列发现其5’端缺失较严重。因此，再次设计YADE的接头引物(5’-ctg gcc gtc caa gac gc-3’，SEQ ID No.13)和(5’-cggtag gat ccc gca gaa c-3’，SEQ ID No.14)和YADE特异引物(5’-cct gag gag ggt ttaact cct gt-3’，SEQ ID No.15)和(5’-gat ctc cct ctt acg ggt tgc act gtc-3’，SEQID No.16)，PCR扩增产物送擎科生物科技有限公司进行序列测定，获得的序列进行拼接，最终获得了抗菌蛋白编码序列如SEQ ID No.17，其氨基酸序列如SEQ ID No.18所示。
NCBI(http://blast.ncbi.nlm.nih.gov/Blast.cgi)数据库进行序列比对，结果显示该抗菌蛋白基因属于I类几丁质酶，因此将该基因命名为BpChiI。
PCR扩增程序
(1)简并引物扩增BpChiI部分序列的程序
98℃3min；98℃10s，65℃10s(每两循环降2℃直至56℃)，72℃30s；98℃10s，56℃10s，72℃30s(25个循环)；72℃5min。
(2)5’-RAC的扩增程序
98℃3min；98℃10S，68℃10s(每两循环降2℃直至62℃),72℃30s；98℃10s，60℃10s，72℃30s(25循环)；72℃5min。
(3)3’-RAC的扩增程序
98℃3min；98℃10S，68℃10s(每一个循环降1℃直至57℃),72℃30s；98℃10s，56℃10s，72℃30s(25循环)；72℃5min。
(4)YADE扩增程序
线性扩增：98℃3min；98℃10s，56℃10s，72℃30s(30个循环)；72℃5min。
指数扩增：98℃3min；98℃10s，67℃10s(每两循环降1℃直至58℃)；72℃30s；98℃10s，57℃15s，72℃30s(25个循环)；72℃5min。
3、构树抗菌蛋白基因BpChiI全长序列的获得
根据上述拼接序列获得的CDS全长设计扩增BpChiI编码区序列的引物(5’-tccccc ggg atg cta ttt cac aaa cata aaa cca aca aat c-3’，SEQ ID No.19)和(5’-cggggt acc tca acc aaa agg cgt ctg gt-3’，SEQ ID No.20)。以总RNA为模板合成的cDNA为模板，扩增BpChiI编码序列全长。
20μL扩增体系包括：模板cDNA，1μL；上下游引物各1μL；PrimerSTAR Max Premix(2X)10μL；ddH2O 7μL。
扩增程序：98℃3min；98℃10s，62℃10s，72℃20s 35个循环；72℃5min。
上述PCR扩增片段克隆到pZERO-Blunt载体，转化大肠杆菌后送擎科生物科技有限公司测序验证，保存序列正确的克隆pZERO-BpChiI。
实施例4、pLGN-35S-BpChiI植物表达载体的构建及工程菌的获得
提取pZERO-BpChiI大肠杆菌的质粒，进行SmaI和KpnI双酶切，回收BpChiI片段，同时用SmaI和KpnI酶切pLGN-35S-Nos质粒，回收大片段。然后用T4 DNA连接酶连接回收的BpChiI片段和pLGN-35S片段，连接产物转化大肠杆菌DH5α，筛选阳性克隆，进行菌落PCR验证以及酶切验证，获得pLGN-35S-BpChiI植物表达载体，该载体具有图4的结构特征。
利用电转化法，将上述获得的pLGN-35S-BpChiI植物表达载体质粒分别转入农杆菌LBA4404和GV3101感受态细胞，利用抗生素筛选标记基因进行抗性筛选，获得阳性克隆，提取农杆菌质粒并用SmaI和KpnI进行双酶切验证，获得含有pLGN-35S-BpChiI植物表达载体的农杆菌工程菌菌株。
实施例5、拟南芥的遗传转化、转基因拟南芥的筛选及转录表达水平分析
1、拟南芥的遗传转化
参照Steven J.Clough and Andrew F.Bent(1998)的浸花转化法，以哥仑比亚野生型拟南芥为材料进行遗传转化，种子成熟后收获农杆菌浸染后的种子。
2、转基因拟南芥植株的筛选
浸花转化法进行遗传转化后收获的种子用75％的酒精灭菌15min，然后均接种于附加100mg/L Km的筛选平板上萌芽，若长成的幼苗为绿色即为转基因植株，待植株2叶以上时移栽入培养拟南芥的专用土壤中(草碳土:蛭石:珍珠岩为3:1:1)，成苗收获种子。每一株植株即为一个转化子。
拟南芥筛选培养基：MS无机+MS有机+Km 100mg/L+2.5g/L Gelrite(固化剂)，pH6.0
3、转基因拟南芥植株RNA的提取及cDNA的合成
以转基因植株幼嫩叶片为材料，按照实施例3的方法进行转基因拟南芥RNA的提取和cDNA的合成。
4、转基因拟南芥中BpChiI基因转录表达水平分析
利用Real-time PCR方法检测转基因拟南芥中BpChiI基因的转录表达水平。
以cDNA为模板扩增BpChiI基因的特异片段。BpChiI基因的上下游引物分别为BpChiI UP：5’-tgacaccacagtcgcctaag-3’(SEQ ID NO.21)和BpChiI DN：5’-gcgtctggttgtagcagtca-3’(SEQ ID NO.22)。以拟南芥AtACT2基因为内标。AtACT2基因的上下游引物分别AtACT2 UP：5’-tatcgctgaccgtatgag-3’(SEQ ID NO.23)和AtACT2 DN：5’-ctgagggaagcaagaatg-3’(SEQ ID NO.24)。
20μL Real-time PCR反应体系包括：cDNA模板1μL，目的基因上下游引物各1μL，2×iQ SYBR Green Supermix 10μL，ddH2O 7μL。
Real-time PCR扩增条件：95℃3min；94℃10s，57℃30s，72℃30s，共扩增40个循环。扩增完成后利用Gene Study软件分析BpChiI基因相对表达量。
Real-time PCR结果表明(图5，A)，转基因拟南芥植株内BpChiI基因都能有效进行转录表达，获得的植株为BpChiI转基因植株。
实施例6、BpChiI转基因拟南芥对黄萎病的抗性
1、转基因拟南芥抗病鉴定接种用黄萎病菌的制备
挑取少许固体PD培养基(马铃薯培养基)保存的落叶型黄萎病菌V991菌株，接种入液体PD培养基，180rpm，26℃振荡培养7d，再按10％(菌液/PD培养基)的比例接种入液体PD培养基，180rpm，26℃振荡培养10d，用四层无菌纱布过滤去除菌液中的菌丝及杂质，去离子水调整孢子浓度达到108个/ml作为接种菌液。
2、转基因拟南芥抗病鉴定接种方法
培养20天的拟南芥幼苗连根拔起，然后带土整齐的摆放入150mm培养皿内，倒入混匀的接种菌液，接种剂量为10mL/株，室温浸泡接种24小时后，再移栽入湿润的土壤中，16小时光照，8小时暗培养，20℃(暗培养)-24℃(光照培养)，湿度70％的光照培养箱内培养。接种2周后，按0-4级的标准统计植株病级并计算病情指数。以转基因株系分离的非转基因植株为对照。
植株病级分级标准：0级：植株叶片无病症；1级：0-25％叶片出现病症；2级：25％-50％叶片出现病症；3级：50％-75％叶片出现病症；4级：75％以上的叶片出现病症。
叶片病级分级标准：0级：叶片无病症；1级：0-25％叶片面积出现病症；2级：25％-50％叶片面积出现病症；3级：50％-75％叶片面积出现病症；4级：75％以上的叶片面积出现病症。
病情指数的计算公式：
病情指数＝(∑〖病级数×植株数〗)/(4×接种植株总数)×100
3、BpChiI基因提高拟南芥对黄萎病的抗性
拟南芥植株接种黄萎病菌14天，统计所有植株每叶的病级。结果显示，非转基因对照(分离自转基因株系)0级叶片的比率为35.98％，BpChiI-2，BpChiI-5，BpChiI-6andBpChiI-7转基因株系的则分别为56.25％,55.85％,59.78％和62.01％(图5，B)，即转基因植株没有病症的叶片比率明显高于非转基因对照，最低株系都高20个百分点。非转基因植株对照的病情指数为47.96，BpChiI-2，BpChiI-5，BpChiI-6和BpChiI-7株系则分别为31.09，23.79，22.72和20.64(图5，C)，转基因株系的病情指数极显著低于非转基因对照。进一步检测转基因拟南芥叶柄内相对黄萎病菌数量，结果显示，非转基因对照内病原菌数量远远低于转基因株系(图5，D)。植株病症显示，接种黄萎病菌14天，野生型植株叶片多数出现了病症，而转基因植株只是基部个别叶片出现了病症(图5，E)。结果表明，BpChiI基因可有效提高拟南芥对黄萎病的抗性。
实施例7、烟草的遗传转化及转基因烟草的获得
1、烟草遗传转化用组织培养培养基
种子萌发培养基：MSB(MS无机盐+B5有机)+1.0％琼脂粉，自来水配制，自然pH。
遗传转化共培养培养基：MSB(MS无机盐+B5有机)+2mg/L NAA+0.5mg/L 6-BA+200μmol/L AS,pH5.6,固体培养基添加1.0％琼脂粉进行固化。
愈伤诱导培养基：MSB(MS无机盐+B5有机)+2mg/L NAA+0.5mg/L 6-BA+1.0％琼脂粉，pH5.8。
幼芽诱导培养基：MSB(MS无机盐+B5有机)+2mg/L 6-BA+1.0％琼脂粉,pH5.8。
生根培养基：MSB(MS无机盐+B5有机)+1.0％琼脂粉，pH6.0。
2、烟草的遗传转化
将含pLGN-35S-BpChiI植物表达载体的重组农杆菌接种入液体YEB培养基，28℃、200rpm振荡培养过夜至OD600 1.0～1.2。菌液离心后收集菌体，并用等体积MSB液体培养基重悬菌体，重悬液即为转化用浸染液。
培养20d的烟草无菌苗叶片，切成3-5mm介方的叶盘，于浸染液内浸染1hr，去除菌液，然后将叶盘接种于共培养培养基，24℃暗培养2天。共培养完成后，外植体继代入附加100mg/L卡那霉素和200mg/L头孢霉素的愈伤诱导培养基，25℃、16hr光照/8hr暗培养的光周期培养，20天后继代入幼芽诱导培养基，之后20天继代一次，至叶盘边缘产生幼芽，将幼芽切下继代入生根培养基生根成苗，幼苗生长至3-4叶移栽入花盆做进一步的分析。
3、BpChiI转基因烟草的获得和分子鉴定
转基因植株的GUS组织化学染色
GUS染色液：500mg/L X-Gluc，0.1mol/L K3Fe(CN)6，0.1mol/L K4Fe(CN)6，1％Triton X-100(V/V)，0.01mol/L Na2EDTA，0.1mol/L磷酸缓冲液(pH7.0)。
pLGN-35S-BpChiI植物表达载体含有35S启动子控制的GUS基因，因此，转基因植株首先可以利用GUS组织化学染色法进行快速鉴定。参照Jefferson(1987)的方法剪取Km抗性幼苗的叶片组织少许，加入GUS组织化学染色液中，37℃染色5h，然后95％乙醇脱色，至绿色去净。最后出现蓝色的为转基因植株，否则为非转基因植株。
4、BpChiI转录表达水平分析
BpChiI转基因烟草植株分别以幼嫩叶片为材料，分别提取GUS阳性和转基因株系分离的非转基因植株叶片的RNA，按cDNA一链合成试剂盒说明书合成各样品RNA的一链cDNA，然后以cDNA为模板扩增BpChiI基因的特异片段。BpChiI基因的上下游引物分别为BpChiI UP：5’-tgacaccacagtcgcctaag-3’(SEQ ID NO.21)和BpChiI DN：5’-gcgtctggttgtagcagtca-3’(SEQ ID NO.22)。以烟草18S基因为内标，18S基因的上下游引物分别18S UP:5’-aggaattgacggaagggca-3’(SEQ ID NO.25)和18S DN:5’-gtgcggcccagaacatctaag-3’(SEQ ID NO.26)。
20μL Real-time PCR反应体系包括：cDNA模板1μL，目的基因上下游引物各1μL，2×iQ SYBR Green Supermix 10μL，ddH2O 7μL。
Real-time PCR扩增条件：95℃3min；94℃10s，57℃30s，72℃30s，共扩增40个循环。扩增完成后利用Gene Study软件分析BpChiI基因相对表达量。
Real-time PCR结果表明(图6，A)，转基因烟草植株内BpChiI基因都能有效进行转录表达，而非转基因植株叶片内都没有检测到该基因的表达。
实施例8、BpChiI转基因烟草对黄萎病的抗性
1、转基因烟草抗病鉴定接种用黄萎病菌的制备
转基因烟草抗病鉴定接种用黄萎病菌的制备方法与实施实例6一致，孢子浓度仍调整为108个孢子/mL。
2、转基因烟草抗病鉴定接种方法
3-4片真叶的烟草植株，从土壤中拔出，用制备的接种液浸根过夜，然后再移栽入营养钵内，之后16小时光照/8小时暗培养，20℃(暗培养)/26℃光照的培养箱内培养，接种14天，按0-4级的5级标准统计叶片的病级并计算病情指数。以转基因株系分离的植株为对照。
植株病级分级标准：0级:叶片无病症；1级:0-25％叶片面积出现病症；2级:25％-50％叶片面积出现病症；3级:50％-75％叶片面积出现病症；4级:75％以上的叶片面积出现病症。
叶片病级的分级标准同实施例6。
病情指数的计算公式：
病情指数＝(∑〖病级数×植株数〗)/(4×接种植株总数)×100。
3、BpChiI基因提高烟草对黄萎病的抗性
接种黄萎病菌14天，BpChiI-2,BpChiI-5,BpChiI-9,BpChiI-13,BpChiI-14和BpChiI-22株系0级和1级叶片比率分别为：87.18％,80.00％,85.00％,74.36％,82.05％and 72.50％，而对照只有20.55％(图6，B)，转基因株系0级和1级的比率较对照提高50％以上。病情指数统计结果显示，接种14天，非转基因对照的病情指数为73.33,转基因株系BpChiI-2,BpChiI-5,BpChiI-9,BpChiI-13,BpChiI-14和BpChiI-22的病情指数分别为12.50,19.27,17.68,17.98,19.49和20.83，与非转基相比，病情指数下降50以上(图6，C)。植株病症显示，接种黄萎病菌14天，野生型植株叶片多数萎蔫或失绿变黄，而转基因植株只是基部叶片出现了病症(图6，D)。结果表明，BpChiI基因可有效提高烟草对黄萎病的抗性。
实施例9、BpChiI提高棉花对黄萎病的抗性
1、瞬时表达BpChiI棉花子叶的获得及黄萎病菌的接种
分别将pLGN-35S-BpChiI载体和pLGN-35S-BE空载的GV3101农杆菌用YEB培养基(含50mg/L Km和50mg/L Rif)活化，在28℃摇床，200rpm培养14-18小时；随后，吸取200μL第一次活化菌液至50mL YEB液体培养基中，28℃/200rpm培养过夜(18-20h)，直至菌液OD600为0.8-1.2，随后加入AS，使终浓度为200μM，再振荡培养30min。随后，将菌液于5000rpm，离心10min，收集菌体，加入等体积瞬时表达重悬液(10mM MES+10mM MgCl2+200μmol/L AS)重悬农杆菌GV3101，室温避光放置3h。
以生长7-10d的冀棉14品种完全展开的棉花子叶为受体材料，使用1mL无针头注射器向棉花子叶下表皮注射上述制备的农杆菌GV3101重悬液，注射完成后，用毛笔在子叶下表皮涂抹黄萎病菌V991孢子悬浮液(108个孢子/mL)。
2、瞬时表达BpChiI提高棉花对黄萎病的抗性
瞬时表达BpChiI的棉花子叶接种黄萎病菌5天，统计子叶的病级，计算不同病级的百分率及病情指数。结果显示，瞬时表达BpChiI的子叶0级和1级百分比分别为16.67％和29.76％，而注射空载农杆菌对照子叶0级和1级百分比则分别为0.00％和19.12％(图7，A)。瞬时表达BpChiI的病情数为40.86，对照的为73.17(图7，B)。结果显示，瞬时表达BpChiI子叶的病级和病情指数均显著下降。瞬时表达BpChiI子叶仅注射孔小范围内出现变黄或坏死症状，而对照则基本上整个子叶都变黄，坏死斑明显大于瞬时表达BpChiI的子叶(图7，C)。结果表明，瞬时表达BpChiI可明显提高棉花对黄萎病的抗性。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
序列表
<110> 西南大学
<120> 构树抗菌蛋白ChiI及其重组表达载体和提高植物对黄萎病抗性中的应用
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<212> DNA
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agtactcaat atccatgcgc tactggcaag caatactatg gccgtggtcc cattcaactc 600
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Tyr Gly Arg Gly Pro Ile Gln Leu Ser Trp Asn Tyr Asn Tyr Gly Pro
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gtgcggccca gaacatctaa g 21
Claims (10)
1.构树抗菌蛋白BpChiI，其特征在于：所述构树抗菌蛋白BpChiI的氨基酸序列如SEQID NO.18所示。
2.权利要求1所述构树抗菌蛋白BpChiI的基因，其特征在于：编码所述构树抗菌蛋白BpChiI的核苷酸序列如SEQ ID NO.17所示，或SEQ ID NO.17所示核苷酸经过一个或几个碱基的取代且编码相同氨基酸的核苷酸序列。
3.含有编码权利要求1所述构树抗菌蛋白BpChiI的基因的重组表达载体。
4.根据权利要求3所述的重组表达载体，其特征在于：所述重组表达载体由SEQ IDNO.17所示序列连入pLGN-35S-Nos质粒的SmaI和KpnI酶切位点处而得。
5.权利要求1所述构树抗菌蛋白BpChiI、权利要求2所述构树抗菌蛋白BpChiI的基因或权利要求3~4任一项所述重组表达载体在提高植物对黄萎病抗性中的应用。
6.根据权利要求5所述的应用，其特征在于：所述植物为拟南芥、烟草或棉花。
7.提高植物对黄萎病抗性的方法，其特征在于：将编码权利要求1所述构树抗菌蛋白BpChiI的基因在植物中过表达，获得对黄萎病具有抗性的植物。
8.根据权利要求7所述提高植物对黄萎病抗性的方法，其特征在于：所述在植物中过表达的方法将编码构树抗菌蛋白BpChiI的基因构建成重组表达载体，然后通过农杆菌介导获得转基因植物。
9.根据权利要求8所述提高植物对黄萎病抗性的方法，其特征在于：所述重组表达载体由SEQ ID NO.17所示序列连入pLGN-35S-Nos质粒的SmaI和KpnI酶切位点处而得。
10.根据权利要求7~9任一项所述提高植物对黄萎病抗性的方法，其特征在于：所述植物为拟南芥、烟草或棉花。
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