苹果MdBAK1s基因表达载体构建、愈伤转化与苹果轮纹病抗性鉴定

发布时间：2018-03-23 04:24

本文选题：苹果　切入点：MdBAK1s　出处：《山东农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：BAK1是植物中重要的一类激酶受体。拟南芥中的研究表明:BAK1介导植物油菜素内酯信号转导、先天性免疫反应以及病原菌致毒蛋白对植物的毒性。苹果中的MdBAK1s与拟南芥中的BAK1具有很高的相似性,而目前国内外有关BAK1在苹果抵抗重要病害中的作用及其在生长发育中的作用研究很少,为此,本研究中,我们克隆了苹果中BAK1的2个同源基因,分别命名为:MdBAK1-1、MdBAK1-2,并对其进行氨基酸序列分析。分别对愈伤组织超表达后生长量的变化,对BR处理的影响,抗病性及抑制表达后对MAPK的信号响应等问题展开了相关研究,这将为进一步研究MdBAK1s在苹果生长发育以及抗病中的作用奠定基础。主要研究结果如下:1.以嘎啦组培苗为材料克隆获得苹果中BAK1的两个同源基因,分别命名为:MdBAK1-1、MdBAK1-2,氨基酸序列分析表明:MdBAK1-1、MdBAK1-2与拟南芥BAK1的序列高度保守,相似度分别为83%、84%,其中胞内激酶区域的保守性更高,均为90%。并且都具有信号肽区域、亮氨酸拉链区域、5个亮氨酸富含区域、脯氨酸富含区、跨膜区以及胞内激酶区。2.获得了稳定超表达MdBAK1s的转基因苹果愈伤组织与转基因苹果株系,并初步观察其表型特征,发现转基因苹果植株长势较弱,部分叶片会随着生长发黄。3.应用RNA干扰的方法抑制苹果中MdBAK1s基因的表达,并获得MdBAK1s表达受抑制的转基因愈伤组织,初步观察其表型特征,发现表达受抑制的转基因愈伤组织颜色与对照相比较浅。4.研究了超表达MdBAK1s的转基因苹果愈伤组织与对照愈伤组织在不同时期生长量的差异,结果表明生长15 d后超表达MdBAK1-1的转基因苹果愈伤组织的生长量明显高于对照,是对照生长量的2.3倍。5.研究了在不同浓度BR处理下超表达MdBAK1s的转基因苹果愈伤组织与对照愈伤组织生长量的差异,结果表明当BR浓度为1.0 nmol/L时可促进苹果愈伤的生长,随着BR浓度的不断增加会抑制苹果愈伤的生长,但当BR浓度为1000 nmol/L时,超表达MdBAK1s的转基因苹果愈伤组织的生长量比普通愈伤明显减少。6.研究了超表达MdBAK1s的转基因苹果愈伤组织与对照愈伤组织对轮纹病菌的抗性,结果表明超表达MdBAK1s的转基因苹果愈伤组织更易感病。7.研究了MdBAK1s表达受抑制的转基因苹果愈伤组织与对照愈伤组织在flg22处理下响应flg22信号的指示性蛋白MAPK6的表达差异,结果表明MdBAK1s表达受抑制后,MAPK6的表达在本底水平上与对照相比变弱。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S436.611

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