本文選題：大白菜VQ蛋白 + 生物信息學(xué)��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：大白菜(Brassica rapa L.ssp.Pekinensis)起源于中國,是我國的特產(chǎn)蔬菜。大白菜富含多種營養(yǎng)物質(zhì),深受消費(fèi)者喜愛。葉球是大白菜食用的最主要器官。大白菜各器官的生長發(fā)育直接影響葉球的發(fā)育,進(jìn)而影響大白菜的產(chǎn)量和品質(zhì)。大白菜的發(fā)育受基因和環(huán)境雙重調(diào)控。VQ蛋白是一類與生長發(fā)育以及響應(yīng)外界環(huán)境脅迫等功能相關(guān)的植物特異性轉(zhuǎn)錄調(diào)控輔助因子,它因含有2個(gè)高度保守的二肽(纈氨酸和谷氨酰胺,VQ)而得名。近年來的研究顯示,VQ蛋白不僅參與種子、下胚軸、花、葉等器官的生長發(fā)育,而且還參與對干旱、鹽、溫度以及病原菌的脅迫應(yīng)答。目前,人們已分離出擬南芥、水稻、大豆、葡萄和玉米的VQ基因并研究了它們的功能,但是在白菜作物中尚未見到相關(guān)報(bào)道。本研究對大白菜的VQ基因家族進(jìn)行了全基因組鑒定及功能分析,取得了一些重要進(jìn)展。1.大白菜VQ基因家族生物信息學(xué)分析以已公布的VQ保守結(jié)構(gòu)域的氨基酸序列以及大白菜數(shù)據(jù)庫(Brassica database)為基礎(chǔ),共鑒定出57個(gè)大白菜VQ成員;基因結(jié)構(gòu)分析發(fā)現(xiàn),57個(gè)BrVQs的編碼區(qū)長度從282bp到1707bp,超過90%的BrVQs無內(nèi)含子;蛋白特征分析發(fā)現(xiàn),BrVQs蛋白序列長度從93aa到568aa,分子量從10.4到63.0千道爾頓,等電點(diǎn)從4.67到10.53;其氨基酸序列僅在VQ結(jié)構(gòu)域處高度保守,而其他位置的序列則相對多變;根據(jù)VQ結(jié)構(gòu)域中L和G位置氨基酸殘基的差異被分成6種類型(LTG、FTG、VTG、LTS、LTV和YTG);另外,除了1個(gè)BrVQ基因以外,其他56個(gè)BrVQs基因非均一地分布在10條染色體上;共線性分析顯示53個(gè)BrVQs分別分布在祖先的7個(gè)t PCK染色體上的13個(gè)blocks區(qū)域,其中41個(gè)BrVQs發(fā)生了片段復(fù)制,2個(gè)BrVQs發(fā)生串聯(lián)復(fù)制;系統(tǒng)進(jìn)化樹顯示BrVQs與AtVQs具有相似的進(jìn)化進(jìn)程,而與Os VQs相異。2.大白菜VQ基因表達(dá)模式分析分別提取大白菜根、短縮莖、老葉、幼葉、花和花蕾的RNA并對57個(gè)BrVQs進(jìn)行各組織表達(dá)模式分析,結(jié)果顯示,26個(gè)BrVQs在根系中表達(dá)較高,13個(gè)BrVQs在老葉中表達(dá)量較高;7個(gè)BrVQs在花蕾中表達(dá)量較高;4個(gè)BrVQs主要在幼葉中有較高的表達(dá)量;3個(gè)BrVQs在短縮莖中被檢測到較高的表達(dá)量;只有1個(gè)BrVQ的表達(dá)量在花中比其他組織高;另外,一些BrVQ同源基因在組織中具有相似的表達(dá)趨勢,而一些則相反。BrVQs在不同非生物脅迫和激素條件下的表達(dá)趨勢顯示,15、11、29、11個(gè)BrVQs分別受PEG6000、Na Cl、35℃和4℃誘導(dǎo)表達(dá);18、25、23個(gè)BrVQs分別受GA3、ABA和SA誘導(dǎo)表達(dá)。大白菜與擬南芥同源VQ基因的表達(dá)模式比較發(fā)現(xiàn),同源基因之間的表達(dá)趨勢既存在相似的也有相異的。以上結(jié)果顯示BrVQ基因可能參與調(diào)控大白菜生長發(fā)育及其抗逆性。3.擬南芥VQ3基因的克隆和功能分析擬南芥和大白菜同屬于十字花科植物,對模式植物擬南芥VQ基因功能的研究將有助于更好地理解大白菜VQ基因功能。目前VQ3基因在擬南芥和大白菜中均未見報(bào)道,所以我們首先對擬南芥VQ3/At1G21326基因進(jìn)來了研究。結(jié)果顯示,AtVQ3基因序列全長720bp,無內(nèi)含子,蛋白序列長度為239aa。表達(dá)模式分析顯示AtVQ3基因在不同組織中具有與BrVQ3-1基因相似的表達(dá)趨勢,都在根部表達(dá)較高;另外,除了ABA處理下無明顯變化外,AtVQ3基因的表達(dá)在PEG6000、Na Cl、35℃、4℃和GA3條件下受到不同程度的誘導(dǎo),尤其在Na Cl處理下上調(diào)表達(dá)最高,這與BrVQ3-1基因的表達(dá)趨勢有所不同。轉(zhuǎn)基因植株表型分析發(fā)現(xiàn)AtVQ3抑制植物生長發(fā)育,主要表現(xiàn)在根系、下胚軸、葉片、開花時(shí)間等;另外,AtVQ3過量表達(dá)植株在不同濃度的鹽脅迫下具有較低的種子萌發(fā)率和較差的幼苗生長情況,而干擾植株則相反。以上結(jié)果說明AtVQ3基因可能在擬南芥生長發(fā)育及其抗鹽性方面具有負(fù)調(diào)控作用。4.野生型Col、35S:AtVQ3-8和AtVQ3-RNAi-3植株轉(zhuǎn)錄組分析為了更加全面地理解AtVQ3基因?qū)M南芥生長發(fā)育及其耐鹽性的影響,對野生型Col、35S:AtVQ3-8和AtVQ3-RNAi-3植株進(jìn)行了RNA-Seq測序。結(jié)果顯示,差異表達(dá)基因在Col vs.35S:AtVQ3-8對比組中共有94個(gè)基因上調(diào)和50個(gè)基因下調(diào),在Col vs.AtVQ3-RNAi-3對比組共有57個(gè)基因上調(diào)和48個(gè)基因下調(diào),而在AtVQ3-RNAi-3 vs.35S:AtVQ3-8對比組共有134個(gè)基因上調(diào)和137個(gè)基因下調(diào);通過對差異表達(dá)基因的深入分析后發(fā)現(xiàn),在35S:AtVQ3-8植株中,明顯上調(diào)表達(dá)的基因包括4個(gè)生長素早期響應(yīng)SAUR基因、油菜素類固醇信號途徑中相關(guān)基因BZS1和RALF23以及生長相關(guān)轉(zhuǎn)錄因子MIF3和LBD38;而細(xì)胞壁合成相關(guān)基因EXPs和EXTs、1個(gè)SAUR基因、植物開花相關(guān)基因ELF4、SOC1和JAC1、鈣調(diào)蛋白相關(guān)基因以及干旱和鹽害響應(yīng)基因GOLS2、ATNCED3和ATCIPK6顯著下調(diào)表達(dá);與35S:AtVQ3-8植株相比,在AtVQ3-RNAi-3植株中上述基因都有明顯相反的表達(dá)趨勢。以上結(jié)果表明:VQ3基因是通過調(diào)控多個(gè)代謝途徑、多個(gè)基因的表達(dá)來參與擬南芥的生長發(fā)育及耐鹽作用。5.大白菜VQ3-1基因的克隆及功能分析為了解BrVQ3-1基因的生物學(xué)功能,我們構(gòu)建了BrVQ3-1過量表達(dá)載體并獲得了擬南芥BrVQ3-1過量表達(dá)植株,結(jié)果顯示,在擬南芥生長發(fā)育方面,BrVQ3-1基因具有與AtVQ3基因相似的功能;但在植物抗鹽性方面,BrVQ3-1基因?qū)}不敏感;另外,我們從AtVQ3轉(zhuǎn)錄組中選取了9個(gè)基因并利用q RT-PCR技術(shù)檢測了它們在35S:BrVQ3-1植株中的表達(dá)情況,結(jié)果顯示5個(gè)基因(EXPA8、EXT3、SAUR31、BZS1和ELF4)在35S:BrVQ3-1植株中表現(xiàn)出明顯的下調(diào)趨勢,這與在35S:AtVQ3-8植株的結(jié)果相似,進(jìn)一步說明了AtVQ3和BrVQ3-1在調(diào)控植物生長發(fā)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.

【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S634.1;Q943.2

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