CN117264972A - 小麦广谱抗病基因及其应用 - Google Patents
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Abstract
本发明属于基因工程技术领域，涉及小麦广谱抗病基因及其应用。本发明采用农杆菌介导的遗传转化方法，获得能够过表达小麦几丁质受体基因TaCEBiP的转基因小麦材料，经接种禾谷镰孢PH‑1、假禾谷镰孢Fp22‑2和条锈菌CYR32发现，过表达TaCEBiP基因小麦对赤霉病、茎基腐病和条锈病的抗病性显著提高。另外，通过比较本发明提供的转基因小麦和野生型小麦的分蘖数、株高、旗叶长、穗长和千粒重数据，证明过表达所述TaCEBiP基因不影响小麦的农艺性状。本发明在小麦农艺形状未受影响的前提下，小麦抗病性显著提高。本发明证实了过表达TaCEBiP基因可应用于创制抗小麦赤霉病、茎基腐病和条锈病种质材料。
Description
技术领域
本发明属于基因工程技术领域，涉及小麦广谱抗病基因及其应用。
背景技术
植物病原真菌严重威胁粮食安全和自然生态系统，其中产生真菌毒素或促使食物变质的真菌进一步降低了食品安全性。稻瘟病菌、灰葡萄孢、柄锈菌属、禾谷镰刀菌、尖孢镰刀菌、布氏白粉菌、禾生球腔菌、炭疽菌属、玉蜀黍黑粉菌和亚麻锈菌是粮食作物中常见的病原真菌，这些真菌的爆发对作物产量和品质影响巨大。
目前，从基因工程角度出发培育持久和广谱抗病品种是防控粮食作物病害最安全有效的措施，随着转基因和基因编辑技术的发展，植物病原真菌的致病机理以及病原-植物互作机理解析为靶向培育抗病品种提供了重要理论基础，病原-植物互作机理导向的抗病品种培育将突破传统育种限制，为作物抗病育种提供重要支持。
植物在长期的进化过程中，产生了一套独特的免疫系统，这种免疫系统能够识别植物病原菌并激发植物免疫反应，进而抵抗植物病原真菌的侵染。目前观点认为，植物免疫系统由病原相关分子模式触发的免疫（Pattern-Triggered Immunity，PTI）和效应蛋白触发的免疫（Effector-Triggered Immunity，ETI）两层免疫系统构成。
其中，PTI是由位于植物细胞膜上的模式识别受体识别微生物或病原相关分子模式（microbial-pathogen-associated molecular pattern，PAMP）或损伤相关分子模式（damage-associated molecular patterns，DAMP）而激发的免疫反应。模式识别受体包括类受体蛋白激酶（RLK）和类受体蛋白（RLP），类受体蛋白激酶含有结合配体的胞外域、跨膜域和胞内激酶域；类受体蛋白除了不含胞内激酶域外，其余结构域与类受体蛋白激酶相似。模式识别受体的胞外域包含不同的基序，例如富亮氨酸重复基序（LRR）、细胞溶解酶基序（Lysin motif，LysM）、凝集素基序（Lectin）、富半胱氨酸重复结构域（CRR）、细胞壁相关激酶结构域（WAK）等基序或结构域。
PTI激活后可以激发一系列细胞生理反应，如钙离子内流、质膜去极化和细胞外碱化、质外体活性氧迸发、一氧化氮的产生、磷脂酸的产生、气孔关闭、肌动蛋白重塑、抗菌化合物和植物激素（水杨酸、茉莉酸和乙烯等）的产生、胼胝质沉积、胞间连丝关闭等，这些生化反应与植物免疫正、负反馈调控系统的协同作用使PTI反应达到顶峰，这些免疫反应共同参与植物对病原菌的广谱抗病性。
几丁质是由β-1,4连接的N-乙酰葡糖胺聚合形成的不溶性多糖，是真菌细胞壁中的重要组分。几丁质的晶体性质赋予其强度和刚性，使其在植物病原菌的侵染结构或侵染菌丝形成过程中发挥关键作用。尽管几丁质可以保护病原菌，使病原菌菌丝在寄主体内生长，然而，作为真菌中保守存在的分子，几丁质也可以作为PAMP被植物几丁质受体识别，并触发植物免疫反应，抵抗真菌的侵染。
基于上述抗病机理，利用基因工程技术调控几丁质受体蛋白基因的表达水平理论上将有利于提高粮食作物的广谱抗病性。然而，目前对于几丁质受体蛋白基因表达水平的调控研究较少，调控其表达能否起到调节植物的广谱抗病性的作用也尚未可知。
发明内容
一方面，传统农药滥用给环境造成巨大的负担；另一方面，几丁质受体蛋白基因表达的调控对于作物广谱抗病性的影响尚缺乏相关机制的研究。基于此，从基因工程角度出发，研究几丁质受体蛋白基因表达水平对作物抗病性的影响将对抗病品种的培育具有重要的贡献价值。具体地，本发明采用如下技术方案：
本发明提供小麦几丁质受体基因TaCEBiP在培育抗赤霉病、茎基腐病和条锈病小麦品种中的应用，具体地，在小麦材料中过表达所述小麦几丁质受体基因TaCEBiP，所小麦几丁质受体基因TaCEBiP的开放阅读框具有如SEQ ID NO:1，或SEQ ID NO:2，或SEQ ID NO:3所示的核苷酸序列。
进一步地，在上述应用中，所述小麦几丁质受体基因TaCEBiP在小麦基因组中有3个拷贝，分别位于小麦基因组的4B染色体、4D染色体和5A染色体上；
位于小麦基因组4B染色体上的TaCEBiP基因开放阅读框具有如SEQ ID NO:1所示的核苷酸序列；
位于小麦基因组4D染色体上的TaCEBiP基因开放阅读框具有如SEQ ID NO:2所示的核苷酸序列；
位于小麦基因组5A染色体上的TaCEBiP基因开放阅读框具有如SEQ ID NO:3所示的核苷酸序列。
进一步地，在上述应用中，所述小麦几丁质受体基因TaCEBiP编码小麦几丁质受体蛋白TaCEBiP；
所述小麦几丁质受体蛋白TaCEBiP由SEQ ID NO:1所示的核苷酸序列编码，并具有如SEQ ID NO:4所示的氨基酸序列；
或所述小麦几丁质受体蛋白TaCEBiP由SEQ ID NO:2所示的核苷酸序列编码，并具有如SEQ ID NO:5所示的氨基酸序列；
或所述小麦几丁质受体蛋白TaCEBiP由SEQ ID NO:3所示的核苷酸序列编码，并具有如SEQ ID NO:6所示的氨基酸序列。
与现有技术相比，本发明“小麦广谱抗病基因及其应用”具有以下有益效果：
本发明采用农杆菌介导的遗传转化方法获得转基因小麦材料，过表达TaCEBiP基因，所述TaCEBiP基因编码小麦几丁质受体蛋白（即TaCEBiP蛋白），经接种禾谷镰孢PH-1、假禾谷镰孢Fp22-2和条锈菌CYR32发现，过表达TaCEBiP基因的小麦对赤霉病、茎基腐病和条锈病的抗病性显著提高。另外，通过比较本发明提供的转基因小麦和野生型小麦的分蘖数、株高、旗叶长、穗长和千粒重数据，证明过表达TaCEBiP基因不影响小麦的主要农艺性状。
本发明提供的转基因小麦，在主要农艺形状未受影响的前提下，抗病性显著提高。另一方面，本发明为小麦抗病品种的培育提供技术途径，同时为小麦真菌病害的防治提供一种新方法，进而帮助减少农药使用造成的病原抗药性和环境污染。
附图说明
图1是位于小麦基因组4B染色体上的TaCEBiP基因表达谱分析示意图。“TPM”表示“transcripts per million reads”，是一个基因表达水平的评估指标。
图2是位于小麦基因组4D染色体上的TaCEBiP基因表达谱分析示意图。“TPM”表示“transcripts per million reads”，是一个基因表达水平的评估指标。
图3是位于小麦基因组5A染色体上的TaCEBiP基因表达谱分析示意图。“TPM”表示“transcripts per million reads”，是一个基因表达水平的评估指标。
图4是TaCEBiP基因过表达载体结构示意图。
图5是TaCEBiP基因表达的电泳结果图。“M”表示Marker；“TaCEBiP-OE1”、“TaCEBiP-OE2”、“TaCEBiP-OE3”、“TaCEBiP-OE5”、“TaCEBiP-OE9”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示能够表达TaCEBiP基因的不同植株；“Plasmid”表示空载体，作为对照组；“Fielder”表示普通的Fielder小麦植株。
图6是“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”的相对表达量柱状图。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
图7是过表达TaCEBiP基因的不同植株在接种禾谷镰孢后的表型结果和发病小穗数统计结果。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
图8是过表达TaCEBiP基因的不同植株在接种假禾谷镰孢后的表型结果和病情指数统计结果。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
图9是过表达TaCEBiP基因的不同植株在接种条锈菌后的表型结果和病情指数统计结果。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
图10是过表达TaCEBiP基因的不同植株的生长情况。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
图11是过表达TaCEBiP基因的不同植株的穗长。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
图12是过表达TaCEBiP基因的不同植株的分蘖数。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
图13是过表达TaCEBiP基因的不同植株的株高。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
图14是过表达TaCEBiP基因的不同植株的旗叶长。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
图15是过表达TaCEBiP基因的不同植株的千粒重。“Fielder”表示普通的Fielder小麦植株；“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”分别表示过表达TaCEBiP基因的不同植株。
具体实施方式
下面结合实施例对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明植物优选单子叶植物，特别优选小麦、水稻、玉米、高粱等作物，以下是以小麦为例的TaCEBiP基因过表达情况。根据TaCEBiP基因CDS区序列，设计能够扩增TaCEBiP基因全长和构建过表达载体的引物，上游引物和下游引物的序列分别见表1。
表1.用于扩增TaCEBiP基因及构建过表达载体的引物序列
以存在于4B染色体的TaCEBiP基因为模板，使用Phanta Max Super-Fidelity DNAPolymerase（南京诺唯赞生物科技股份有限公司）试剂盒，参照试剂盒说明书加入如表2所示的反应体系，经PCR获得TaCEBiP基因扩增产物。
表2.TaCEBiP基因PCR反应体系
扩增程序为：95℃ 3min；95℃ 15s，55℃ 15s，72℃ 1min，35次循环；72℃ 5min；16℃保温。
将获得的TaCEBiP基因PCR产物进行凝胶电泳，并回收目标片段，利用Gateway®BP Clonase™ II enzyme mix（Invitrogen）将TaCEBiP基因构建到pDONR中间载体（克隆载体），进而利用同样的Gateway克隆技术将TaCEBiP克隆到小麦表达载体pANIC-6E上，构建成功后，将构建的重组载体pANIC-6E-TaCEBiP转入农杆菌EHA105菌株，TaCEBiP基因过表达载体结构示意图见图4所示。
以Fielder小麦品种作为受体，通过农杆菌介导的小麦遗传转化法，对小麦未成熟的幼胚进行转化，经分化、筛选、再生和生根后，最终获得转基因小麦植株。
将转基因小麦植株种植到土壤基质中，之后剪取叶片提取DNA，通过PCR和电泳检测是否含有目的基因片段，电泳结果见图5。图5中可见，“TaCEBiP-OE1”、“TaCEBiP-OE2”、“TaCEBiP-OE3”、“TaCEBiP-OE5”、“TaCEBiP-OE9”、“TaCEBiP-OE11”和“TaCEBiP-OE19”小麦植株内均有目的基因的表达。
对上述含有TaCEBiP基因的植株，提取RNA并反转录获得cDNA，利用荧光定量PCR（qRT-PCR）检测植株中TaCEBiP基因的相对表达量，筛选出过表达TaCEBiP基因的小麦植株。本发明获得能够过表达TaCEBiP基因的植株有：“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”，具体的相对表达量见图6。
对过表达TaCEBiP基因的小麦植株和野生型小麦植株做以下几方面检测：
1）选择对Fielder小麦品种致病力强的禾谷镰孢野生型菌株PH-1，通过单花滴注法分别将其接种至上述过表达TaCEBiP基因的Fielder小麦植株（“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”）和野生型Fielder小麦植株，比较两者对赤霉病的抗性。实验结果见图7，图7是过表达TaCEBiP基因的不同植株在接种禾谷镰PH-1孢后的表型结果和发病小穗数统计结果。由图7可知，与野生型Fielder小麦植株相比，过表达TaCEBiP基因的Fielder小麦植株在接种禾谷镰孢野生型菌株PH-1 14d后发病穗数明显减少。剥出小麦籽粒后发现，野生型Fielder小麦大部分的籽粒已经发病，而过表达TaCEBiP基因的Fielder小麦仅有少数籽粒发病，表明过表达TaCEBiP基因能够显著增强小麦植株对赤霉病的抗性。
2）选择对Fielder小麦品种致病力强的假禾谷镰孢Fp22-2，分别将其接种至上述过表达TaCEBiP基因的Fielder小麦植株（“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”）和野生型Fielder小麦植株，比较两者对茎基腐病的抗性。实验结果见图8，图8是过表达TaCEBiP基因的不同植株在接种假禾谷镰孢Fp22-2后的表型结果和病情指数统计结果。由图8可知，与野生型Fielder小麦植株相比，过表达TaCEBiP基因的Fielder小麦植株在接种假禾谷镰孢菌株Fp22-2后基腐病的病情严重度显著降低，表明过表达TaCEBiP基因能够显著增强小麦植株对基腐病的抗性。
3）选择对Fielder小麦品种致病力强的条锈菌CYR32，分别将其接种至上述过表达TaCEBiP基因的Fielder小麦植株（“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”）和野生型Fielder小麦植株，比较两者对条锈病的抗性。实验结果见图9，图9是过表达TaCEBiP基因的不同植株在接种条锈菌后的表型结果和病情指数统计结果。由图9可知，与野生型Fielder小麦植株相比，过表达TaCEBiP基因的Fielder小麦植株在接种条锈菌CYR32后，条锈病的病情严重度显著降低，表明过表达TaCEBiP基因能够显著增强小麦植株对条锈病的抗性。
4）比较过表达TaCEBiP基因的Fielder小麦植株（“TaCEBiP-OE2”、“TaCEBiP-OE11”和“TaCEBiP-OE19”）和野生型Fielder小麦植株的主要农艺性状（包括分蘖数、株高、旗叶长、穗长和千粒重），明确过表达TaCEBiP基因对主要农艺性状的影响情况。实验结果见图10~图15，图10~图15分别是过表达TaCEBiP基因的不同植株的生长情况、穗长、分蘖数、株高、旗叶长、千粒重的统计结果。由图10~图15可知，过表达TaCEBiP基因不影响小麦主要农艺性状。
以上所述的实施例只是本发明的一部分实施例，而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。在依据本发明构思的条件下本领域普通技术人员进行的相关推演和替换，在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
Claims (3)
1.小麦几丁质受体基因TaCEBiP在培育抗赤霉病、茎基腐病和条锈病小麦品种中的应用，其特征在于，在小麦材料中过表达所述小麦几丁质受体基因TaCEBiP，所小麦几丁质受体基因TaCEBiP的开放阅读框具有如SEQ ID NO:1，或SEQ ID NO:2，或SEQ ID NO:3所示的核苷酸序列。
2.根据权利要求1所述的应用，其特征在于，所述小麦几丁质受体基因TaCEBiP在小麦基因组中有3个拷贝，分别位于小麦基因组的4B染色体、4D染色体和5A染色体上；
位于小麦基因组4B染色体上的TaCEBiP基因开放阅读框具有如SEQ ID NO:1所示的核苷酸序列；
位于小麦基因组4D染色体上的TaCEBiP基因开放阅读框具有如SEQ ID NO:2所示的核苷酸序列；
位于小麦基因组5A染色体上的TaCEBiP基因开放阅读框具有如SEQ ID NO:3所示的核苷酸序列。
3.根据权利要求1所述的应用，其特征在于，所述小麦几丁质受体基因TaCEBiP编码小麦几丁质受体蛋白TaCEBiP；
所述小麦几丁质受体蛋白TaCEBiP由SEQ ID NO:1所示的核苷酸序列编码，并具有如SEQ ID NO:4所示的氨基酸序列；
或所述小麦几丁质受体蛋白TaCEBiP由SEQ ID NO:2所示的核苷酸序列编码，并具有如SEQ ID NO:5所示的氨基酸序列；
或所述小麦几丁质受体蛋白TaCEBiP由SEQ ID NO:3所示的核苷酸序列编码，并具有如SEQ ID NO:6所示的氨基酸序列。
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