大白菜现蕾前叶色转变过程中蛋白组学分析及BrTUAs鉴定

发布时间：2018-06-22 21:21

本文选题：大白菜 + 抽薹　；参考：《东北农业大学》2016年博士论文

【摘要】：大白菜〔Brassica campestris L.ssp.pekinensis(Lour)Olsson〕起源于我国,是十字花科芸薹属中重要的蔬菜作物之一,是我国各类蔬菜中栽培面积最大,栽培历史悠久的一种蔬菜作物。大白菜因其具有产量高、生产成本低、种植方法简单、耐贮运强,是我国北方城乡人民的“当家菜”,在菜篮子工程中具有重要的地位。近年来,大白菜反季节栽培生产发展迅速,春季大白菜的种植面积不断扩大,但由于栽培管理不当及气候因素的影响,常有先期抽薹现象的发生。先期抽薹是指叶球尚未充分形成之前发生的抽薹现象,叶球失去商品价值和食用价值,导致大幅度减产绝收,给生产造成重大损失。在我国春季及高海拔地区的夏白菜生产中,先期抽薹常常成为困扰生产的一道难题。同时,在种子生产、加代繁殖过程中,经常遇到不抽薹或花期延后现象,严重影响种子产量和质量。因此研究大白菜开花的分子遗传机理,尤其是研究抽薹相关基因调控,对于大白菜良种繁育和合理栽培调控具有重要意义。我们在育种实践中发现,大白菜在花芽分化后、抽薹前表现出一个叶色转变过程,叶片颜色从鲜亮的绿色转为没有光泽的暗灰色后花茎就会伸长、抽薹,叶色转变似乎是大白菜抽薹的一个生理信号。前期分析了激素、色素含量变化与叶色转变的关系及叶色转变过程中AP1、LEFY、FT等基因的表达变化。本试验力图在已有的基础上,通过叶色转变过程蛋白质组学分析,并对相关基因表达进行进一步的鉴定,加深对大白菜发育分子生物学机制的认识,以期为大白菜抽薹开花分子调控提供更多的理论依据。本试验取得如下结论:(1)试验以C30自交系为试验材料,利用双向电泳技术比较大白菜叶色转变前后植株生长点部位蛋白组分变化,发现叶色转变前后特异表达蛋白点35个,差异表达蛋白点32个。.对17个边界清晰、表达量较高的蛋白点进行MALDI-TOF/MS质谱技术分析,鉴定出4个有信息价值的差异蛋白点,分别为V型H+转运ATP酶E1亚基、Rubisco的大亚基、S-腺苷甲硫氨酸合成酶和微管蛋白α-2。(2)用目标蛋白推测的核苷酸序列在大白菜基因组数据库blast分析后设计特异引物,荧光定量PCR分析表明BrVHA-E1、BrSAMS、BrrbcL和BrTUA6在大白菜叶色转变前后表达发生明显变化,并与蛋白组学分析结果一致,可能参与了抽薹调控和发育。(3)以拟南芥6个TUA基因氨基酸序列作为查询种子序列,在大白菜基因组数据库中在线运行Blastp,共发现12个BrTUA。.通过对12个BrTUA基因结构、进化和功能预测,发现BrTUA基因长度、内含子、外显子和启动子区域伴随基因组进化发生了较大变化;12个BrTUA中仅有5个编码完整的α-微管蛋白。(4)TUA系统发育分析,表明BrTUA与AtTUA、BnTUA亲缘关系较近,TUA的进化与物种进化同步。(5)通过对5个编码完整的α-微管蛋白的BrTUA启动子区域顺式作用元件分析,在起始密码子上游1.5 kb区域,共发现27种类型、超过60个顺式作用元件。BrTUA启动子区域有大量光周期反应元件;不同BrTUA中低温反应原件、激素反应相关元件差异较大;5个BrTUA启动子区域都有一个或多个组织特异性表达元件。推测不同的α-tubulin在大白菜生长发育调控中扮演者不同角色。(6)5个编码完整的α-微管蛋白的BrTUA呈现出时空表达的差异;尤其在春化前后、现蕾前的叶色转变过程中,BrTUA表达差异较大,说明BrTUA参与了大白菜抽薹开花调控。(7)通过叶色转变前施用外源物质对大白菜抽薹开花的影响及BrTUA表达分析,确定春化后、叶色转变前,向大白菜生长点喷施微管敏感药物紫杉醇及GA3和MJ,可调控BrTUA表达并实现对抽薹开花的调控。
[Abstract]:Chinese Cabbage [Brassica campestris L.ssp.pekinensis (Lour) Olsson], originated from China, is one of the most important vegetable crops in the Brassica genus of Cruciferae. It is one of the largest vegetable crops in our country and has a long history of cultivation. Chinese cabbage has high yield, low production cost, simple planting method and strong storage and transportation. The "dish" of the people in urban and rural areas in the north of China has an important position in the vegetable basket project. In recent years, the production and development of Chinese cabbage in the anti season cultivation and development are rapid, and the planting area of Chinese cabbage is expanding in spring. However, because of improper cultivation and the influence of climate factors, the phenomenon of bolting in Chang Youxian period. The bolting phenomenon occurred before the full formation, the leaf ball lost its commodity value and edible value, resulting in a great loss of yield and loss of production. In the production of Chinese cabbage in spring and high altitude areas in China, the early bolting is often a difficult problem to perplex production. It is important to study the molecular genetic mechanism of flowering of Chinese cabbage, especially to study the regulation of bolting related genes, which is of great significance to the breeding and rational cultivation and regulation of Chinese cabbage. After a leaf color transformation process, the leaf color from bright green to dark grey was extended, bolting and leaf color change seemed to be a physiological signal of bolting in Chinese cabbage. The relationship between the changes of the hormone, the change of pigment content and leaf color transformation and the expression of AP1, LEFY, FT in the process of leaf color transformation were analyzed. On the basis of the present experiment, we try to analyze the proteomic analysis of leaf color transformation process and further identify the related gene expression to deepen the understanding of the molecular biological mechanism of Chinese cabbage development so as to provide more theoretical basis for the regulation of bolting and flowering molecules in Chinese cabbage. The results are as follows: (1) the experiment is C3 0 the self inbred lines were used as experimental materials to compare the changes in the protein components of the plant growth points before and after the leaf color transformation of Chinese cabbage, and found that there were 35 specific protein points before and after the leaf color transformation, 32 differentially expressed protein points, and 17 distinct boundaries and high expression levels were analyzed by MALDI-TOF/MS mass spectrometry, and 4 of the protein points were identified. The difference protein points with information value are V type H+ transshipment ATP enzyme E1 subunit, Rubisco large subunit, S- adenosine methionine synthetase and microtubulin alpha -2. (2), and the specific nucleotide sequences are designed by the target protein in the genome database blast analysis of Chinese cabbage, and the fluorescence quantitative PCR analysis shows BrVHA-E1, BrSAMS, BrrbcL and. The expression of BrTUA6 before and after the leaf color transformation of Chinese cabbage was obviously changed, and was consistent with the results of proteomics analysis. (3) the 6 TUA gene amino acid sequences of Arabidopsis were used as the query seed sequence, and Blastp was transported online in the Chinese cabbage genome database. A total of 12 BrTUA.. Were found to pass to 12 BrTUA bases. Due to structural, evolutionary and functional prediction, the BrTUA gene length, introns, exons and promoter regions have been accompanied by great changes in genome evolution; only 5 intact alpha microtubules are encoded in the 12 BrTUA. (4) TUA phylogenetic analysis shows that the relationship between BrTUA and AtTUA, BnTUA is close, and the evolution of TUA is synchronized with the evolution of species. (5) through (5) After the analysis of cis acting elements in the BrTUA promoter region of 5 intact alpha microtubules, 27 types were found in the 1.5 KB region upstream of the starting codon, and a large number of photoperiod reaction elements were found in the.BrTUA promoter region of more than 60 cis acting elements; the hormone reaction related elements in different BrTUA were different; 5 There are one or more tissue specific expression elements in the BrTUA promoter region. It is speculated that different alpha -tubulin plays different roles in the growth and development regulation of Chinese cabbage. (6) the difference in temporal and spatial expression of the 5 encoding complete alpha microtubule BrTUA shows the difference in temporal and spatial expression, especially in the process of the leaf color transformation before and before the vernalization. The expression of BrTUA is poor. BrTUA participated in the regulation of bolting and flowering of Chinese cabbage. (7) the effect of exogenous substances on bolting and flowering of Chinese cabbage before leaf color transformation and analysis of BrTUA expression, after vernalization, before leaf color transformation, microtubule sensitive drug paclitaxel and GA3 and MJ were sprayed to the growth point of Chinese cabbage, and BrTUA expression could be regulated and bolting flowering was realized. Control.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S634.1

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