菌根诱导的柑橘Papain家族基因的挖掘及表达特性分析

发布时间：2018-02-28 23:27

本文关键词： 柑橘丛枝菌根木瓜蛋白酶丛枝降解苜蓿　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：柑橘根毛稀少,主要通过与丛枝菌根真菌(AMF,arbuscular mycorrhizal fungis)形成共生来吸收营养。丛枝菌根真菌不仅可以帮助柑橘根系吸收养分,还可以增强其抵抗生物与非生物胁迫的能力,因此开展柑橘菌根共生方面的研究意义重大。目前对柑橘菌根的研究主要停留在生理水平,柑橘与菌根互作在分子水平的研究鲜有报道。本课题组前期研究发现5个柑橘木瓜蛋白酶(Papain)基因受到菌根的诱导表达,暗示该类基因与菌根共生有关。基于此,本研究首先通过进化分析筛选关键的柑橘Papain基因,然后利用苜蓿模式系统分析了这些基因在菌根形成和发育过程中的时空表达、蛋白质的亚细胞定位以及基因功能,主要结果如下:通过Papain基因的系统进化分析,我们发现4个柑橘基因(PtrCP1、PtrCP2、PtrCP4、PtrCP5)、9个苜蓿基因和3个水稻基因聚集在一个不含非菌根植物(拟南芥)基因的进化枝,且该进化枝的全部柑橘、苜蓿基因均受到丛枝菌根的强烈诱导;柑橘Papain基因PtrCP3则存在于另一个独立分支。通过qRT-PCR检测表明,PtrCP1、PtrCP2、PtrCP3、PtrCP4、PtrCP5在菌根中的表达量相比未接种对照极显著上调(p0.001),暗示其参与菌根共生形成的调控;利用苜蓿毛根瞬时转化体系研究了柑橘Papain基因在菌根中的表达特性:启动子-GUS分析表明,PtrCP1、PtrCP3、Ptr CP4的启动子在丛枝细胞中受到特异性诱导,而残缺的PtrCP2启动子则未表现组织特异性;融合表达载体Native promoter-PtrCP4-GFP主要在丛枝部位尤其是丛枝的尖端富集表达。为探讨Papain基因在菌根中的功能,我们选择了与柑橘PtrCP1、PtrCP2、PtrCP4、PtrCP5基因在同一分支的苜蓿基因MtCP1和MtCP9,通过筛选鉴定获得了mtcp1和mtcp9的纯合Tnt突变株系,正用于后续表型检测。鉴于Papain蛋白的水解酶特性及其在根瘤中促进共生体衰老的功能,本研究的结果表明,柑橘Papain基因PtrCP1、PtrCP2、PtrCP3、PtrCP4、PtrCP5也很可能通过发挥其水解酶活性来调节菌根的发育和衰老,从而参与菌根共生的分子调控。
[Abstract]:Citrus root hairs are rare and absorb nutrients mainly by symbiosis with arbuscular mycorrhizal fungis of arbuscular mycorrhizal fungi. Arbuscular mycorrhizal fungi can not only help citrus roots absorb nutrients, but also enhance their ability to resist biological and abiotic stresses. Therefore, the research on citrus mycorrhizal symbiosis is of great significance. There are few reports on the interaction between citrus and mycorrhizal mycorrhizae at molecular level. Our previous study found that five papain genes were induced by mycorrhizal mycorrhizal expression, suggesting that these genes are related to mycorrhizal symbiosis. In this study, the key citrus Papain genes were screened by evolutionary analysis, and then the temporal and spatial expression of these genes during mycorrhizal formation and development, the subcellular localization of proteins and the gene function were systematically analyzed by alfalfa model. The main results are as follows: through phylogenetic analysis of Papain gene, we found that 4 citrus genes PtrCP1, PtrCP2, PtrCP4, PtrCP5, 9 alfalfa genes and 3 rice genes were clustered in an evolutionary branch of a non-mycorrhizal plant (Arabidopsis thaliana) gene. All citrus and alfalfa genes were strongly induced by arbuscular mycorrhiza. PtrCP3 of citrus Papain gene existed in another independent branch. QRT-PCR detection showed that PtrCP1, PtrCP2, PtrCP3, PtrCP4, PtrCP4, PtrCP5 were significantly up-regulated in mycorrhizal mycorrhizae, implying that PtrCP1 was involved in the regulation of mycorrhizal symbiosis. The expression characteristics of citrus Papain gene in mycorrhiza were studied by using the transient transformation system of alfalfa hairy root. The promoter -GUS analysis showed that the promoter of PtrCP1, PtrCP3, PtrCP3, PTR CP4 was specifically induced in the arbuscular cells, but the incomplete PtrCP2 promoter showed no tissue specificity. The fusion expression vector Native promoter-PtrCP4-GFP was mainly expressed in the arbuscular site, especially in the tip of the arbuscular branch. In order to investigate the function of Papain gene in mycorrhizal cells, The homozygous Tnt mutant lines of mtcp1 and mtcp9 were selected from alfalfa genes MtCP1 and MtCP9 in the same branch of PtrCP1, PtrCP2, PtrCP4 and PtrCP5 genes of Citrus, and the homozygous Tnt mutants of mtcp1 and mtcp9 were obtained. In view of the hydrolase properties of Papain protein and its function in promoting the senescence of symbionts in nodule, the results of this study indicate that, The citrus Papain gene PtrCP1, PtrCP2, PtrCP3, PtrCP4 and PtrCP5 may also regulate mycorrhizal development and senescence by exerting its hydrolase activity, thus participating in the molecular regulation of mycorrhizal symbiosis.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S666

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