小麦白粉菌应答小分子RNA的分离、鉴定及功能分析
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摘要
MicroRNA（miRNA）是一类具有调控作用的非编码小分子RNA，其长度多在20至24nt之间。miRNA通常于转录后水平对基因表达起调控作用，它在植物的生长发育过程中发挥着关键性的作用。同时，越来越多的证据表明寄主小分子RNA在植物免疫反应的过程中同样起着重要的作用。小麦是重要的农作物，具有很高的经济价值，虽然小麦中克隆的miRNA越来越多,但人们对其在小麦白粉病抵抗过程中的作用却知道不多。研究miRNA在白粉菌抵抗过程中的作用十分重要，揭示其机理对于提高小麦白粉病抗病育种的效率有着积极的作用。热激蛋白9（0Hsp90）是真核生物和原核生物中普遍存在的分子伴侣，它在信号传导、蛋白质折叠、蛋白降解以及生长发育方面都发挥着重要的作用。细胞质Hsp90在植物体对病原菌的抵抗过程中有重要的作用，高等植物中效应子触发的免疫（effector-triggered immunity，ETI）是由细胞内免疫受体R蛋白介导的，许多R蛋白的功能发挥是在细胞质Hsp90协助下完成的。
本研究以用高通量测序的方法发掘白粉菌响应小分子RNA，发现在白粉菌侵染条件下有38个显著表达差异的miRNA，其中包括22个已知和16个新的miRNA。其中，miR396的测序结果用Northern杂交得到了很好的重复，所以我们对其进行了一系列的功能分析：
⑴为了发掘小麦中响应白粉菌侵染的miRNA，我们对白粉菌处理前后小麦幼叶构建的小分子RNA文库进行高通量测序，并用Northern杂交的方法对其中的部分miRNA的应答进行验证，两种检测方法下miR396的响应模式一致。
⑵为了验证小麦中miRNA的靶基因，我们构建了小麦PmA/北京837和北京837两叶期幼苗经白粉菌处理后多个时间点混合样品的degradome文库，共检测到了232个miRNA的靶基因。这是小麦中首次采用Degradome测序的方法分析miRNA靶基因。
⑶Degradome测序的结果显示小麦中miR396的靶基因之一是Hsp90，5’RACE证实miR396可以切割TaHsp90.2和TaHsp90.3的mRNA，证明TaHsp90.2和TaHsp90.3确实是miR396的靶基因。
⑷为了分析小麦3类Hsp90基因的功能，我们采用了病毒诱导的基因沉默系统，发现TaHsp90.2和TaHsp90.3在小麦白粉病抗性途径中也发挥重要作用。
本研究通过对白粉菌接种前后的小分子RNA文库进行深度测序分析，发掘响应白粉菌的miRNA。通过病毒介导的基因沉默系统对其靶基因进行功能分析发现，两个Hsp90基因参与小麦白粉病抵抗。结合Hsp90在小麦条锈抗性途径中作用的研究，本研究拓展了这两个基因的在抗病过程中的功能，揭示了一条miRNA介导小麦抵抗白粉菌的作用机制。
MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between20and24nucleotides. They play a key role during plant development by negatively regulating gene expression atthe posttranscriptional level. Moreover, increasing evidence indicates that host small RNAs also play acritical role during the plant immune responses. Wheat is one of the most important crops with higheconomic value. More and more miRNAs have been discovered in wheat, but functions of thesemiRNAs on resistance to powdery mildew (PM) have rarely been disclosed. It is important to study themiRNAs roles in PM resistance which may facilitate more efficient strategy in wheat breeding forresistance to PM. Heat shock proteins90s (Hsp90s) are ubiquitous molecular chaperones in the cells ofeukaryta and eubacteria. They play key roles in signal transduction, protein folding, protein degradation,as well as growth and developmental programs. Cytosolic Hsp90proteins have also been found to playimportant roles in plant immune responses to pathogens. Pathogen effector-triggered immunity (ETI) inhigher plants is known to be mediated by intracellular immune receptors-R proteins. Many R proteinshave been found to be functionally dependent on cytosolic Hsp90s.
In this study, to discover and analyze important small RNAs responsive to PM in wheat, wesequenced the wheat small RNAs libraries before and after Bgt inoculation. A total of38miRNAs wereobtained, including22known and16novel miRNAs. The sequencing results of miR396were repeatedby Northern blot, so we characterized the function of miR396, the main results are as following:
⑴To discover miRNAs response to PM,6small RNA libraries using the young leaf of wheatinoculated with PM were constructed. The expression patterns of some miRNAs are confirmed bynorthern blot, miR396responses the same way under the two detection methods.
⑵To comprehensively investigate small RNA targets, a degradomelibraries derived from themixture tissues of young leaf before and after Bgt inoculation of wheat was constructed and used forsequencing. Two hundreds and thirty-two genes targeted were detected in our degradome libraries. It isthe first time in wheat using degradome sequencing analysis of the target genes of miRNAs.
⑶Degradome sequencing results showed that Hsp90is the target genes of miR396in wheat, andthis result is confirmed using5’ RACE, which confirms the result that TaHsp90.2and TaHsp90.3is thetarget genes of miR396.
⑷To study functions of three classes of wheat Hsp90genes, virus-induced gene silencing system(VIGS) was used to analyze and the result showed that TaHsp90.2and TaHsp90.3also play importantroles in the wheat PM resistance pathway.
Our studies discovered miRNAs responsive to PM by small RNA deep sequencing of librariesderived from tissues inoculated with PM. VIGS assays showed that miR396targets two Hsp genes thatare involved in PM resistance. Combined with previous studies that Hsp90genes play important roles inthe stripe rust resistance pathway of wheat, the function of the these genes and unraveled an miRNAmediated PM resistancec mechanism in wheat were expanded in this study.
本研究以用高通量测序的方法发掘白粉菌响应小分子RNA，发现在白粉菌侵染条件下有38个显著表达差异的miRNA，其中包括22个已知和16个新的miRNA。其中，miR396的测序结果用Northern杂交得到了很好的重复，所以我们对其进行了一系列的功能分析：
⑴为了发掘小麦中响应白粉菌侵染的miRNA，我们对白粉菌处理前后小麦幼叶构建的小分子RNA文库进行高通量测序，并用Northern杂交的方法对其中的部分miRNA的应答进行验证，两种检测方法下miR396的响应模式一致。
⑵为了验证小麦中miRNA的靶基因，我们构建了小麦PmA/北京837和北京837两叶期幼苗经白粉菌处理后多个时间点混合样品的degradome文库，共检测到了232个miRNA的靶基因。这是小麦中首次采用Degradome测序的方法分析miRNA靶基因。
⑶Degradome测序的结果显示小麦中miR396的靶基因之一是Hsp90，5’RACE证实miR396可以切割TaHsp90.2和TaHsp90.3的mRNA，证明TaHsp90.2和TaHsp90.3确实是miR396的靶基因。
⑷为了分析小麦3类Hsp90基因的功能，我们采用了病毒诱导的基因沉默系统，发现TaHsp90.2和TaHsp90.3在小麦白粉病抗性途径中也发挥重要作用。
本研究通过对白粉菌接种前后的小分子RNA文库进行深度测序分析，发掘响应白粉菌的miRNA。通过病毒介导的基因沉默系统对其靶基因进行功能分析发现，两个Hsp90基因参与小麦白粉病抵抗。结合Hsp90在小麦条锈抗性途径中作用的研究，本研究拓展了这两个基因的在抗病过程中的功能，揭示了一条miRNA介导小麦抵抗白粉菌的作用机制。
MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between20and24nucleotides. They play a key role during plant development by negatively regulating gene expression atthe posttranscriptional level. Moreover, increasing evidence indicates that host small RNAs also play acritical role during the plant immune responses. Wheat is one of the most important crops with higheconomic value. More and more miRNAs have been discovered in wheat, but functions of thesemiRNAs on resistance to powdery mildew (PM) have rarely been disclosed. It is important to study themiRNAs roles in PM resistance which may facilitate more efficient strategy in wheat breeding forresistance to PM. Heat shock proteins90s (Hsp90s) are ubiquitous molecular chaperones in the cells ofeukaryta and eubacteria. They play key roles in signal transduction, protein folding, protein degradation,as well as growth and developmental programs. Cytosolic Hsp90proteins have also been found to playimportant roles in plant immune responses to pathogens. Pathogen effector-triggered immunity (ETI) inhigher plants is known to be mediated by intracellular immune receptors-R proteins. Many R proteinshave been found to be functionally dependent on cytosolic Hsp90s.
In this study, to discover and analyze important small RNAs responsive to PM in wheat, wesequenced the wheat small RNAs libraries before and after Bgt inoculation. A total of38miRNAs wereobtained, including22known and16novel miRNAs. The sequencing results of miR396were repeatedby Northern blot, so we characterized the function of miR396, the main results are as following:
⑴To discover miRNAs response to PM,6small RNA libraries using the young leaf of wheatinoculated with PM were constructed. The expression patterns of some miRNAs are confirmed bynorthern blot, miR396responses the same way under the two detection methods.
⑵To comprehensively investigate small RNA targets, a degradomelibraries derived from themixture tissues of young leaf before and after Bgt inoculation of wheat was constructed and used forsequencing. Two hundreds and thirty-two genes targeted were detected in our degradome libraries. It isthe first time in wheat using degradome sequencing analysis of the target genes of miRNAs.
⑶Degradome sequencing results showed that Hsp90is the target genes of miR396in wheat, andthis result is confirmed using5’ RACE, which confirms the result that TaHsp90.2and TaHsp90.3is thetarget genes of miR396.
⑷To study functions of three classes of wheat Hsp90genes, virus-induced gene silencing system(VIGS) was used to analyze and the result showed that TaHsp90.2and TaHsp90.3also play importantroles in the wheat PM resistance pathway.
Our studies discovered miRNAs responsive to PM by small RNA deep sequencing of librariesderived from tissues inoculated with PM. VIGS assays showed that miR396targets two Hsp genes thatare involved in PM resistance. Combined with previous studies that Hsp90genes play important roles inthe stripe rust resistance pathway of wheat, the function of the these genes and unraveled an miRNAmediated PM resistancec mechanism in wheat were expanded in this study.
引文
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