【作者】 张媛媛；

【导师】 栾雨时；

【作者基本信息】 大连理工大学 ， 生物学， 2021， 硕士

【摘要】 番茄(Solanum lycopersicum)易受多种病原菌的侵害,是研究植物患病机理的理想物种。晚疫病是番茄的一种毁灭性病害,发病时会造成严重减产。常规的防治方法存在极大局限性,因此利用基因工程手段发掘有利基因,培育抗病品种尤为重要。基因组上的非编码RNA(Non-coding RNA,nc RNA)在植物对生物胁迫的响应中起着关键作用,它包括微小RNA(Micro RNA,mi RNA)、长非编码RNA(Long non-coding RNA,lnc RNA)、小干扰RNA(Small interfering RNA,si RNA)等多种类型。mi R394属于mi RNA家族成员之一,其表达水平在病原菌侵染后会发生显著变化。实验室前期预测了mi R394的内源竞争性RNA(Competing endogenous RNA,ce RNA):lnc RNA40787,lnc RNA27177和lnc RNA42566。根据晚疫病菌侵染后的表达特性,最终挑选了lnc RNA40787进行下一步研究。随后分别过表达和沉默了mi R394及lnc RNA40787,阐明了mi R394与lnc RNA40787在番茄抗晚疫病中的作用机制及功能。首先,为探究mi R394在番茄抗晚疫病中起到的作用,本研究通过与植物高效表达载体连接以及短串联靶标模拟技术(Short tandem target mimic,STTM)构建了mi R394的过表达及沉默载体。利用瞬时侵染将两种重组载体转入番茄,发现mi R394的过表达会降低其靶基因LCR(Leaf Curling Responsiveness,LCR)的转录水平,影响茉莉酸(Jasmonic acid,JA)信号通路的关键基因表达,最终降低番茄对晚疫病感染的抵抗力;沉默后结果则与之相反。证明了mi R394的负调控作用。其次,在获得了mi R394相关的三个lnc RNAs(lnc RNA40787,lnc RNA27177和lnc RNA42566)的序列后,分析了这三个lnc RNAs上存在的内源靶标模拟(Endogenous target mimicry,e TM)位点。接种晚疫病菌后对mi R394、三个lnc RNAs、LCR的表达特性进行分析,发现三个lnc RNAs均呈现出明显的上升趋势,mi R394被显著抑制,LCR表达模式则与lnc RNA40787大致相同。以上结果证明三者确实会对该胁迫做出响应。进一步研究发现,在番茄植株瞬时过表达lnc RNA40787后,mi R394表达水平显著下降,LCR基因的表达量则明显上升,番茄对晚疫病的抗性得到了提高。该结果初步证明mi R394可被lnc RNA40787吸附,进而调控靶基因的转录水平,影响番茄对晚疫病的抗性。最后,构建了lnc RNA40787的沉默及突变载体。沉默lnc RNA40787后,mi R394的表达量显著升高,LCR的表达水平下降。接种晚疫病菌后,番茄叶片的病斑面积和病斑直径明显变大,植株抗病能力降低。为进一步说明lnc RNA40787与mi R394二者间的直接作用关系,瞬时转入了lnc RNA40787突变体载体p BI121-mlnc RNA40787,发现mi R394在p BI121-mlnc RNA40787转化叶片中表达量并无显著差异变化。综合以上结果,我们可以得出结论:mi R394的确可以被lnc RNA40787吸附。综上所述,本研究验证了lnc RNA40787是mi R394的ce RNA,明确了mi R394在番茄感染晚疫病后的负调控作用。我们的研究提出了lnc RNA40787-mi R394-LCR调控网络,为揭示番茄抗晚疫病的分子机制提供了理论基础和参考。更多还原

【Abstract】 Tomato(Solanum lycopersicum)is vulnerable to a variety of pathogens,and it is an ideal specie for studying the pathogenic mechanism of plants.Late blight is a devastating disease of tomato,which can cause severe yield reduction.Conventional prevention and control methods have some limitations,so it is particularly important to use genetic engineering methods to discover key genes and cultivate disease-resistant varieties.Non-coding RNA(nc RNA)in the genome plays a crucial role in the response of plants to biological stress.Nc RNA includes micro RNA(mi RNA),long non-coding RNA(lnc RNA),small interfering RNA(si RNA),and many other types.mi R394 is a member of the mi RNA family,and its expression level changed significantly after pathogen infection.Our previous study predicted three lnc RNAs(lnc RNA40787,lnc RNA27177,and lnc RNA42566)that contain mi R394 endogenous target mimics(e TM),which may exist as the competitive endogenous RNAs(ce RNAs)of mi R394.Based on the expression pattern among these lnc RNAs and mi R394,lnc RNA40787 was chosen for further investigation.Subsequently,mi R394 and lnc RNA40787 were overexpressed and silenced,respectively,to clarify the mechanism and function of mi R394 and lnc RNA40787 in tomato resistance against late blight.First,to explore the role of mi R394 in tomato resistance against late blight,our study constructed the overexpression plasmid by ligating it with the efficient plant expression vector p BI121.Short tandem target mimic(STTM)was used to silence mi R394.Two recombinant vectors were transformed into the tomato by transient infection.We found that the overexpression of mi R394 could reduce the transcription level of its target gene Leaf Curling Responsiveness(LCR),and affect the expression level of jasmonic acid(JA)related genes.These ultimately reduced tomato resistance to late blight.The negative regulatory role of the mi R394 was demonstrated.Secondly,after obtaining the sequences of these three lnc RNAs(lnc RNA40787,lnc RNA27177,and lnc RNA42566)that related to mi R394,the e TM sites on these lnc RNAs were analyzed.Furthermore,these lnc RNAs showed a clear upward trend,while mi R394 was significantly inhibited,and the LCR expression pattern was roughly the same as lnc RNA40787.For further exploration,we overexpressed lnc RNA40787.After transiently overexpressed lnc RNA40787,the expression level of mi R394 decreased significantly,and the expression level of LCR increased accordingly.These results proved that mi R394 can be decoyed by lnc RNA40787,thereby regulating the transcription level of target gene and affecting the resistance of tomato to late blight.Finally,the silencing and mutation vector of lnc RNA40787 were constructed.After lnc RNA40787 was silenced,the expression of mi R394 increased significantly,and the expression of LCR decreased.After inoculated with Phytophthora infestans,the diameter of lesion on tomato leaves increased significantly.To illustrate the direct interaction between lnc RNA40787 and mi R394,the mutation vector of lnc RNA40787 was constructed,and it was found that the expression of mi R394 did not change significantly after the transformation of mutant lnc RNA40787.Based on the above results,we can conclude that mi R394 can indeed be decoyed by lnc RNA40787.In summary,this study validated the existence of the ce RNA-lnc RNA40787 of mi R394 and found the negative regulatory effect of mi R394 after the infection of Phytophthora infestans.Our research provides information on the lnc RNA40787-mi R394-LCR regulatory network,providing a theoretical basis and reference for revealing the molecular mechanisms of tomato resistance against late blight.更多还原