本文關(guān)鍵詞： 柑橘 叢枝菌根 木瓜蛋白酶 叢枝降解 苜蓿　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：柑橘根毛稀少,主要通過與叢枝菌根真菌(AMF,arbuscular mycorrhizal fungis)形成共生來吸收營養(yǎng)。叢枝菌根真菌不僅可以幫助柑橘根系吸收養(yǎng)分,還可以增強其抵抗生物與非生物脅迫的能力,因此開展柑橘菌根共生方面的研究意義重大。目前對柑橘菌根的研究主要停留在生理水平,柑橘與菌根互作在分子水平的研究鮮有報道。本課題組前期研究發(fā)現(xiàn)5個柑橘木瓜蛋白酶(Papain)基因受到菌根的誘導(dǎo)表達,暗示該類基因與菌根共生有關(guān)�；诖�,本研究首先通過進化分析篩選關(guān)鍵的柑橘Papain基因,然后利用苜蓿模式系統(tǒng)分析了這些基因在菌根形成和發(fā)育過程中的時空表達、蛋白質(zhì)的亞細(xì)胞定位以及基因功能,主要結(jié)果如下:通過Papain基因的系統(tǒng)進化分析,我們發(fā)現(xiàn)4個柑橘基因(PtrCP1、PtrCP2、PtrCP4、PtrCP5)、9個苜�；蚝�3個水稻基因聚集在一個不含非菌根植物(擬南芥)基因的進化枝,且該進化枝的全部柑橘、苜�；蚓艿絽仓膹娏艺T導(dǎo);柑橘Papain基因PtrCP3則存在于另一個獨立分支。通過qRT-PCR檢測表明,PtrCP1、PtrCP2、PtrCP3、PtrCP4、PtrCP5在菌根中的表達量相比未接種對照極顯著上調(diào)(p0.001),暗示其參與菌根共生形成的調(diào)控;利用苜蓿毛根瞬時轉(zhuǎn)化體系研究了柑橘Papain基因在菌根中的表達特性:啟動子-GUS分析表明,PtrCP1、PtrCP3、Ptr CP4的啟動子在叢枝細(xì)胞中受到特異性誘導(dǎo),而殘缺的PtrCP2啟動子則未表現(xiàn)組織特異性;融合表達載體Native promoter-PtrCP4-GFP主要在叢枝部位尤其是叢枝的尖端富集表達。為探討Papain基因在菌根中的功能,我們選擇了與柑橘PtrCP1、PtrCP2、PtrCP4、PtrCP5基因在同一分支的苜蓿基因MtCP1和MtCP9,通過篩選鑒定獲得了mtcp1和mtcp9的純合Tnt突變株系,正用于后續(xù)表型檢測。鑒于Papain蛋白的水解酶特性及其在根瘤中促進共生體衰老的功能,本研究的結(jié)果表明,柑橘Papain基因PtrCP1、PtrCP2、PtrCP3、PtrCP4、PtrCP5也很可能通過發(fā)揮其水解酶活性來調(diào)節(jié)菌根的發(fā)育和衰老,從而參與菌根共生的分子調(diào)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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S666

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