大白菜VQ基因家族鉴定及功能分析

发布时间：2018-05-11 15:34

本文选题：大白菜VQ蛋白 + 生物信息学　；参考：《甘肃农业大学》2016年博士论文

【摘要】：大白菜(Brassica rapa L.ssp.Pekinensis)起源于中国,是我国的特产蔬菜。大白菜富含多种营养物质,深受消费者喜爱。叶球是大白菜食用的最主要器官。大白菜各器官的生长发育直接影响叶球的发育,进而影响大白菜的产量和品质。大白菜的发育受基因和环境双重调控。VQ蛋白是一类与生长发育以及响应外界环境胁迫等功能相关的植物特异性转录调控辅助因子,它因含有2个高度保守的二肽(缬氨酸和谷氨酰胺,VQ)而得名。近年来的研究显示,VQ蛋白不仅参与种子、下胚轴、花、叶等器官的生长发育,而且还参与对干旱、盐、温度以及病原菌的胁迫应答。目前,人们已分离出拟南芥、水稻、大豆、葡萄和玉米的VQ基因并研究了它们的功能,但是在白菜作物中尚未见到相关报道。本研究对大白菜的VQ基因家族进行了全基因组鉴定及功能分析,取得了一些重要进展。1.大白菜VQ基因家族生物信息学分析以已公布的VQ保守结构域的氨基酸序列以及大白菜数据库(Brassica database)为基础,共鉴定出57个大白菜VQ成员;基因结构分析发现,57个BrVQs的编码区长度从282bp到1707bp,超过90%的BrVQs无内含子;蛋白特征分析发现,BrVQs蛋白序列长度从93aa到568aa,分子量从10.4到63.0千道尔顿,等电点从4.67到10.53;其氨基酸序列仅在VQ结构域处高度保守,而其他位置的序列则相对多变;根据VQ结构域中L和G位置氨基酸残基的差异被分成6种类型(LTG、FTG、VTG、LTS、LTV和YTG);另外,除了1个BrVQ基因以外,其他56个BrVQs基因非均一地分布在10条染色体上;共线性分析显示53个BrVQs分别分布在祖先的7个t PCK染色体上的13个blocks区域,其中41个BrVQs发生了片段复制,2个BrVQs发生串联复制;系统进化树显示BrVQs与AtVQs具有相似的进化进程,而与Os VQs相异。2.大白菜VQ基因表达模式分析分别提取大白菜根、短缩茎、老叶、幼叶、花和花蕾的RNA并对57个BrVQs进行各组织表达模式分析,结果显示,26个BrVQs在根系中表达较高,13个BrVQs在老叶中表达量较高;7个BrVQs在花蕾中表达量较高;4个BrVQs主要在幼叶中有较高的表达量;3个BrVQs在短缩茎中被检测到较高的表达量;只有1个BrVQ的表达量在花中比其他组织高;另外,一些BrVQ同源基因在组织中具有相似的表达趋势,而一些则相反。BrVQs在不同非生物胁迫和激素条件下的表达趋势显示,15、11、29、11个BrVQs分别受PEG6000、Na Cl、35℃和4℃诱导表达;18、25、23个BrVQs分别受GA3、ABA和SA诱导表达。大白菜与拟南芥同源VQ基因的表达模式比较发现,同源基因之间的表达趋势既存在相似的也有相异的。以上结果显示BrVQ基因可能参与调控大白菜生长发育及其抗逆性。3.拟南芥VQ3基因的克隆和功能分析拟南芥和大白菜同属于十字花科植物,对模式植物拟南芥VQ基因功能的研究将有助于更好地理解大白菜VQ基因功能。目前VQ3基因在拟南芥和大白菜中均未见报道,所以我们首先对拟南芥VQ3/At1G21326基因进来了研究。结果显示,AtVQ3基因序列全长720bp,无内含子,蛋白序列长度为239aa。表达模式分析显示AtVQ3基因在不同组织中具有与BrVQ3-1基因相似的表达趋势,都在根部表达较高;另外,除了ABA处理下无明显变化外,AtVQ3基因的表达在PEG6000、Na Cl、35℃、4℃和GA3条件下受到不同程度的诱导,尤其在Na Cl处理下上调表达最高,这与BrVQ3-1基因的表达趋势有所不同。转基因植株表型分析发现AtVQ3抑制植物生长发育,主要表现在根系、下胚轴、叶片、开花时间等;另外,AtVQ3过量表达植株在不同浓度的盐胁迫下具有较低的种子萌发率和较差的幼苗生长情况,而干扰植株则相反。以上结果说明AtVQ3基因可能在拟南芥生长发育及其抗盐性方面具有负调控作用。4.野生型Col、35S:AtVQ3-8和AtVQ3-RNAi-3植株转录组分析为了更加全面地理解AtVQ3基因对拟南芥生长发育及其耐盐性的影响,对野生型Col、35S:AtVQ3-8和AtVQ3-RNAi-3植株进行了RNA-Seq测序。结果显示,差异表达基因在Col vs.35S:AtVQ3-8对比组中共有94个基因上调和50个基因下调,在Col vs.AtVQ3-RNAi-3对比组共有57个基因上调和48个基因下调,而在AtVQ3-RNAi-3 vs.35S:AtVQ3-8对比组共有134个基因上调和137个基因下调;通过对差异表达基因的深入分析后发现,在35S:AtVQ3-8植株中,明显上调表达的基因包括4个生长素早期响应SAUR基因、油菜素类固醇信号途径中相关基因BZS1和RALF23以及生长相关转录因子MIF3和LBD38;而细胞壁合成相关基因EXPs和EXTs、1个SAUR基因、植物开花相关基因ELF4、SOC1和JAC1、钙调蛋白相关基因以及干旱和盐害响应基因GOLS2、ATNCED3和ATCIPK6显著下调表达;与35S:AtVQ3-8植株相比,在AtVQ3-RNAi-3植株中上述基因都有明显相反的表达趋势。以上结果表明:VQ3基因是通过调控多个代谢途径、多个基因的表达来参与拟南芥的生长发育及耐盐作用。5.大白菜VQ3-1基因的克隆及功能分析为了解BrVQ3-1基因的生物学功能,我们构建了BrVQ3-1过量表达载体并获得了拟南芥BrVQ3-1过量表达植株,结果显示,在拟南芥生长发育方面,BrVQ3-1基因具有与AtVQ3基因相似的功能;但在植物抗盐性方面,BrVQ3-1基因对盐不敏感;另外,我们从AtVQ3转录组中选取了9个基因并利用q RT-PCR技术检测了它们在35S:BrVQ3-1植株中的表达情况,结果显示5个基因(EXPA8、EXT3、SAUR31、BZS1和ELF4)在35S:BrVQ3-1植株中表现出明显的下调趋势,这与在35S:AtVQ3-8植株的结果相似,进一步说明了AtVQ3和BrVQ3-1在调控植物生长发育方面具有相似的功能。
[Abstract]:Chinese Cabbage (Brassica rapa L.ssp.Pekinensis) originated from China and is a special vegetable in China. Chinese cabbage is rich in many nutrients and is very popular with consumers. Leaf ball is the most important organ for Chinese cabbage. The growth and development of various organs of Chinese cabbage directly affect the development of leaf ball, and affect the yield and quality of Chinese cabbage. The hair of Chinese Cabbage The dual regulation of gene and environment.VQ protein is a kind of plant specific transcriptional regulator associated with growth and development and response to external environmental stress. It is named after 2 highly conserved two peptides (valine and glutamine, VQ). In recent years, the research shows that VQ protein not only participates in seed, hypocotyl, flower and leaf. At present, people have isolated the VQ genes of Arabidopsis, rice, soybeans, grapes and corn, and studied their functions, but not in the Chinese cabbage crops. This study has carried out a whole gene for the VQ gene family of Chinese cabbage. Group identification and functional analysis, some important progress was made in the bioinformatics analysis of the VQ gene family of Chinese cabbage.1.. Based on the published amino acid sequences of the VQ conserved domain and the Chinese cabbage database (Brassica database), 57 cabbages VQ members were identified. Genetic analysis found that the length of the 57 BrVQs coding regions was from 282bp. To 1707bp, more than 90% of BrVQs without introns; protein characteristics analysis found that BrVQs protein sequence length from 93aa to 568aa, molecular weight from 10.4 to 63 thousand Dalton, isoelectric point from 4.67 to 10.53; its amino acid sequence is only highly conserved at the VQ domain, and the sequence of other locations is relatively changeable; according to the VQ structure domain L and G position amino acid residue The differences in the base were divided into 6 types (LTG, FTG, VTG, LTS, LTV and YTG); in addition, the other 56 BrVQs genes were distributed on 10 chromosomes in addition to the 1 BrVQ genes, and the co linear analysis showed that 53 BrVQs distributed in 13 blocks regions on the 7 T PCK chromosomes of the ancestors, 41 of which were replicated in fragment and 2. The phylogenetic tree showed that BrVQs and AtVQs had similar evolutionary process, and the VQ gene expression pattern analysis of.2. and.2. in.2. of Os VQs extracted the RNA of Chinese cabbage root, the short stem, the old leaf, the young leaf, the flower and the bud and the expression pattern of the 57 BrVQs. The results showed that 26 BrVQs were expressed in the root system. High expression of 13 BrVQs in old leaves; 7 BrVQs in bud expression higher; 4 BrVQs mainly in young leaves have higher expression; 3 BrVQs in the short stem was detected higher expression; only 1 BrVQ expression in the flower higher than the other tissues; in addition, some of the BrVQ homologous genes in the tissues have similar expression. The trend, and some contrary.BrVQs expression trends under different abiotic stress and hormone conditions, showed that 15,11,29,11 BrVQs was induced by PEG6000, Na Cl, 35 and 4 C, 18,25,23 BrVQs was induced by GA3, ABA and SA, respectively. The expression pattern of the homologous genes of Chinese cabbage and Arabidopsis found that the table of homologous genes was found. The trend is similar and different. The results show that the BrVQ gene may be involved in the regulation of the growth and resistance of Chinese cabbage and its resistance to.3., the cloning and functional analysis of the VQ3 gene of Arabidopsis thaliana. The Arabidopsis and Chinese cabbage belong to the cruciferous plants. The study of the VQ gene function of the Arabidopsis thaliana in the pattern plant will help to better understand the whiteness. VQ gene function. At present, VQ3 gene is not reported in Arabidopsis and Chinese cabbage, so we first studied Arabidopsis VQ3/At1G21326 gene. The results showed that the whole length of AtVQ3 gene sequence was 720bp, no intron, and the protein sequence length was 239aa. expression pattern analysis showed that the AtVQ3 gene had the BrVQ3-1 gene in different tissues. In addition, the expression of AtVQ3 gene was induced by different degrees in PEG6000, Na Cl, 35, 4 and GA3, especially under the Na Cl treatment, in addition to ABA treatment. The phenotype analysis of transgenic plants was different. It was found that AtVQ3 inhibited plant growth and development, mainly in root, hypocotyl, leaf and flowering time. In addition, AtVQ3 overexpressed plants had lower seed germination rate and poor seedling growth under different concentration of salt stress, while the interference plants were opposite. The results showed that AtVQ3 gene may grow and develop in Arabidopsis thaliana. Its salt resistance has a negative regulation of.4. wild type Col, 35S:AtVQ3-8 and AtVQ3-RNAi-3 plant transcriptome analysis in order to understand the effect of AtVQ3 gene on the growth and salt tolerance of Arabidopsis thaliana more comprehensively, and the RNA-Seq sequencing of wild type Col, 35S:AtVQ3-8 and AtVQ3-RNAi-3 plants. The results show that the differentially expressed genes are in Col v. There were 94 up-regulated genes and 50 down regulated genes in the s.35S:AtVQ3-8 contrast group. There were 57 up-regulated genes and 48 down regulated genes in the Col vs.AtVQ3-RNAi-3 contrast group, while in the AtVQ3-RNAi-3 vs.35S:AtVQ3-8 comparison group, there were 134 up regulation and 137 gene downregulation. In the plant, the genes that are obviously up-regulated include the early response of 4 auxin to the SAUR gene, the related genes BZS1 and RALF23 in the brassin steroid pathway and the related transcription factors MIF3 and LBD38, while the cell wall synthesis related genes EXPs and EXTs, the 1 SAUR genes, and the flowering related genes ELF4, SOC1 and JAC1, calmodulin related groups GOLS2, ATNCED3 and ATCIPK6 were significantly down regulated because of drought and salt response genes. Compared with 35S:AtVQ3-8 plants, the above genes in AtVQ3-RNAi-3 plants have obvious opposite expression trends. The above results show that the VQ3 gene is involved in the growth and development and tolerance of Arabidopsis by regulating multiple metabolic pathways and the expression of multiple genes. The cloning and functional analysis of VQ3-1 gene of Brassica.5. in salt action in order to understand the biological function of BrVQ3-1 gene, we constructed a BrVQ3-1 overexpression vector and obtained the BrVQ3-1 overexpressed plant of Arabidopsis thaliana. The results showed that the BrVQ3-1 gene had the function similar to the AtVQ3 gene in the growth and development of Arabidopsis, but the salt resistance of the plant was in the plant. On the other hand, the BrVQ3-1 gene was insensitive to salt; in addition, we selected 9 genes from the AtVQ3 transcriptome and detected their expression in 35S:BrVQ3-1 plants by Q RT-PCR technique. The results showed that 5 genes (EXPA8, EXT3, SAUR31, BZS1 and ELF4) showed obvious downward trend in the 35S:BrVQ3-1 plant, which was in 35S:AtVQ3-8 plants. The results are similar, indicating that AtVQ3 and BrVQ3-1 have similar functions in regulating plant growth and development.

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S634.1;Q943.2

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