本文選題：蘋果　切入點(diǎn)：MdBAK1s　出處：《山東農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：BAK1是植物中重要的一類激酶受體。擬南芥中的研究表明:BAK1介導(dǎo)植物油菜素內(nèi)酯信號(hào)轉(zhuǎn)導(dǎo)、先天性免疫反應(yīng)以及病原菌致毒蛋白對(duì)植物的毒性。蘋果中的MdBAK1s與擬南芥中的BAK1具有很高的相似性,而目前國內(nèi)外有關(guān)BAK1在蘋果抵抗重要病害中的作用及其在生長發(fā)育中的作用研究很少,為此,本研究中,我們克隆了蘋果中BAK1的2個(gè)同源基因,分別命名為:MdBAK1-1、MdBAK1-2,并對(duì)其進(jìn)行氨基酸序列分析。分別對(duì)愈傷組織超表達(dá)后生長量的變化,對(duì)BR處理的影響,抗病性及抑制表達(dá)后對(duì)MAPK的信號(hào)響應(yīng)等問題展開了相關(guān)研究,這將為進(jìn)一步研究MdBAK1s在蘋果生長發(fā)育以及抗病中的作用奠定基礎(chǔ)。主要研究結(jié)果如下:1.以嘎啦組培苗為材料克隆獲得蘋果中BAK1的兩個(gè)同源基因,分別命名為:MdBAK1-1、MdBAK1-2,氨基酸序列分析表明:MdBAK1-1、MdBAK1-2與擬南芥BAK1的序列高度保守,相似度分別為83%、84%,其中胞內(nèi)激酶區(qū)域的保守性更高,均為90%。并且都具有信號(hào)肽區(qū)域、亮氨酸拉鏈區(qū)域、5個(gè)亮氨酸富含區(qū)域、脯氨酸富含區(qū)、跨膜區(qū)以及胞內(nèi)激酶區(qū)。2.獲得了穩(wěn)定超表達(dá)MdBAK1s的轉(zhuǎn)基因蘋果愈傷組織與轉(zhuǎn)基因蘋果株系,并初步觀察其表型特征,發(fā)現(xiàn)轉(zhuǎn)基因蘋果植株長勢(shì)較弱,部分葉片會(huì)隨著生長發(fā)黃。3.應(yīng)用RNA干擾的方法抑制蘋果中MdBAK1s基因的表達(dá),并獲得MdBAK1s表達(dá)受抑制的轉(zhuǎn)基因愈傷組織,初步觀察其表型特征,發(fā)現(xiàn)表達(dá)受抑制的轉(zhuǎn)基因愈傷組織顏色與對(duì)照相比較淺。4.研究了超表達(dá)MdBAK1s的轉(zhuǎn)基因蘋果愈傷組織與對(duì)照愈傷組織在不同時(shí)期生長量的差異,結(jié)果表明生長15 d后超表達(dá)MdBAK1-1的轉(zhuǎn)基因蘋果愈傷組織的生長量明顯高于對(duì)照,是對(duì)照生長量的2.3倍。5.研究了在不同濃度BR處理下超表達(dá)MdBAK1s的轉(zhuǎn)基因蘋果愈傷組織與對(duì)照愈傷組織生長量的差異,結(jié)果表明當(dāng)BR濃度為1.0 nmol/L時(shí)可促進(jìn)蘋果愈傷的生長,隨著BR濃度的不斷增加會(huì)抑制蘋果愈傷的生長,但當(dāng)BR濃度為1000 nmol/L時(shí),超表達(dá)MdBAK1s的轉(zhuǎn)基因蘋果愈傷組織的生長量比普通愈傷明顯減少。6.研究了超表達(dá)MdBAK1s的轉(zhuǎn)基因蘋果愈傷組織與對(duì)照愈傷組織對(duì)輪紋病菌的抗性,結(jié)果表明超表達(dá)MdBAK1s的轉(zhuǎn)基因蘋果愈傷組織更易感病。7.研究了MdBAK1s表達(dá)受抑制的轉(zhuǎn)基因蘋果愈傷組織與對(duì)照愈傷組織在flg22處理下響應(yīng)flg22信號(hào)的指示性蛋白MAPK6的表達(dá)差異,結(jié)果表明MdBAK1s表達(dá)受抑制后,MAPK6的表達(dá)在本底水平上與對(duì)照相比變?nèi)酢?br/>[Abstract]:BAK1 is an important kinase receptor in plants. Studies in Arabidopsis thaliana have shown that WBAK1 mediates the signal transduction of vegetable luteinolactone. MdBAK1s in apple and BAK1 in Arabidopsis thaliana have high similarity. However, there are few studies on the role of BAK1 in apple resistance to important diseases and its role in growth and development. Therefore, we cloned two homologous genes of BAK1 in apple. They were named as: MdBAK1-1, MdBAK1-2, and their amino acid sequences were analyzed. The changes of callus growth after overexpression, the effect on Br treatment, the disease resistance and the signal response to MAPK after inhibition of expression were studied. This will lay a foundation for further study on the role of MdBAK1s in apple growth, development and disease resistance. The main results are as follows: 1. Two homologous genes of BAK1 in apple were cloned from Gala plantlets. The amino acid sequence analysis showed that the sequence of MdBAK1-1 and MdBAK1-2 was highly conserved with Arabidopsis thaliana BAK1, and the similarity was 830.The intracellular kinase region was even more conserved, and all had signal peptide regions. Leucine zipper region, leucine rich region, proline rich region, transmembrane region and intracellular kinase region .2. transgenic apple callus and transgenic apple lines with stable overexpression of MdBAK1s were obtained, and their phenotypic characteristics were preliminarily observed. It was found that the growth of transgenic apple plants was weak, and some leaves would turn yellow with growth. The RNA interference method was used to inhibit the expression of MdBAK1s gene in apple, and to obtain the transgenic callus with inhibited MdBAK1s expression. The phenotypic characteristics of the transgenic callus were preliminarily observed. It was found that the color of transgenic callus expressing inhibited was shallower than that of control. The difference of callus growth between transgenic apple callus and control callus with overexpression of MdBAK1s at different stages was studied. The results showed that the callus growth of transgenic apple with overexpression of MdBAK1-1 was significantly higher than that of control after 15 days of growth. The difference of callus growth between transgenic apple and control callus treated with different concentrations of Br was studied. The results showed that when Br concentration was 1.0 nmol/L, the growth of apple callus could be promoted. With the increasing of Br concentration, the growth of apple callus was inhibited, but when Br concentration was 1000 nmol/L, the growth of apple callus was inhibited. The growth of transgenic apple callus with overexpression of MdBAK1s was significantly lower than that of common callus. The resistance of transgenic apple callus with overexpression of MdBAK1s to rotifer was studied. The results showed that transgenic apple callus with overexpression of MdBAK1s was more susceptible to disease. The expression of MAPK6, an indicative protein in response to flg22 signal in flg22 treatment, was studied between transgenic apple callus and control callus. The results showed that the expression of MAPK6 was weaker at the background level than that in the control group after the expression of MdBAK1s was inhibited.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S436.611

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