山葡萄論文：山葡萄兩個(gè)ICE1同源基因的克隆與表達(dá)特性研究

1、 山葡萄論文：山葡萄兩個(gè)ICE1同源基因的克隆與表達(dá)特性研究【中文摘要】低溫是限制植物分布與生長的重要環(huán)境因素,而且低溫凍害是全球農(nóng)林業(yè)普遍存在的自然災(zāi)害,給全世界農(nóng)林業(yè)造成了巨大的損失。隨著分子生物學(xué)技術(shù)的發(fā)展,寒害研究已經(jīng)深入到分子水平,從而促進(jìn)植物抗寒基因工程的發(fā)展。本試驗(yàn)以山葡萄為材料,采用電子克隆技術(shù)從山葡萄EST數(shù)據(jù)庫中拼接到兩個(gè)ICEl基因,應(yīng)用戶RT-PCR擴(kuò)增得到兩個(gè)基因編碼區(qū)全長序列,分別命名為VaICEla和VaICE1b。結(jié)果如下：VaICE1a基因的cDNA全長為1617bp,根據(jù)核苷酸推導(dǎo)出的氨基酸序列分析說明它由538個(gè)氨基酸組成,命名為VaICE1a。cDNA序

2、列和編碼的蛋白序列結(jié)果顯示其含有一個(gè)富s區(qū)、核定位信號區(qū)(NLS)、轉(zhuǎn)膜區(qū)。山葡萄VaICEla基因與歐洲葡萄(XP-002275711)、蘋果、毛果楊、歐洲葡萄(XP-002284528)、擬南芥、小麥的同源性分別為99%、61%、62%、64%、60%、76%。該序列在C端高度保守,具有ICE基因典型的BHLH結(jié)構(gòu)域。系統(tǒng)發(fā)育分析將VaICE1a基因歸入ICEl進(jìn)化系。三級結(jié)構(gòu)分析說明ValCEla為混合型蛋白。上述結(jié)果說明VaICE1a可能為ICEl基因家族的一個(gè)新成員。半定量PCR結(jié)果說明VaICE1a在山葡萄的不同組織部位均不同程度的表達(dá),在四種非生物逆境脅迫下VaICE1a基因能夠

3、誘導(dǎo)表達(dá),與對照組比較表達(dá)量沒有明顯差異。VaICE1b基因的cDNA全長為1653bp,根據(jù)核苷酸推導(dǎo)出的氨基酸序列分析說明它由550個(gè)氨基酸組成,命名為VaICElb。山葡萄VaICE1b基因的cDNA序列和編碼的蛋白序列結(jié)果顯示其含有一個(gè)富s區(qū)、核定位信號區(qū)(NLS)、轉(zhuǎn)膜區(qū)。通過blast分析發(fā)現(xiàn)山葡萄VaICElb基因與歐洲葡萄(XP-002275711)、蘋果、毛果楊、歐洲葡萄(XP-002284528)、擬南芥、小麥的同源性分別為64%、68%、68%、99%、52%、73%。該序列在C端高度保守,具有ICE基因典型的BHLH結(jié)構(gòu)域。系統(tǒng)發(fā)育分析將VaICE1b基因歸入ICE1進(jìn)

4、化系。三級結(jié)構(gòu)分析說明VaICE1b的為混合型蛋白。上述結(jié)果說明VaICE1b可能為ICEl基因家族的一個(gè)新成員。半定量PCR結(jié)果說明VaICE1b在山葡萄的不同組織部位均不同程度的表達(dá),在四種非生物逆境脅迫下VaICE1b基因均能夠誘導(dǎo)表達(dá),與對照組相比表達(dá)量相對較高。成功構(gòu)建了VaICEla和VaICElb基因的植物雙元表達(dá)載體?！居⑽恼縏he low temperature is limiting important environmental factor of geographical dist of plants and growth, moreover the low tem

5、perature freeze injury is the global agriculture forestry universal existence natural disaster, has created the massive loss for the world agriculture forestry. Along with the molecular biology technologys development, the winter-killing research already penetrated into the molecular level, thus pro

6、motion cold resistant of plant genetic engineering development.This experiment take the amur grape as material,Two ICE1 genes were isolated from Vitis amurensis Rupr.by bioformatic analysis and RT-PCR. named as VaICE1a and the VaICE1b gene. The result is as follows:VaICE1a gene cDNA span is 1617bp,

7、according to the nucleotide analysis that it was composed 538 amino acids, named VaICE1a. The cDNA sequence and the code protein sequence showed that it includes a rich s area, the nucleus localization signaling zone (NLS), to transfer the membrane area. Homology analysis showed that the deduced VaI

8、CE1 protein was highly homologous to the Vitis vinifera (XP-002275711)、Malus、Populus trichocarpa、Vitis vinifera (XP-002284528)、Arabidopsis thaliana、Triticum aestivum, the identity are 99%、61%、62%、64%、60%、76%, respectively. Sequence in C terminal is high conserved, and has bHLH typical structure. Phy

9、logenetic analysis also indicated that VaICE1b belongs to the ICE1 gene family. Tertiary Structure analysis that is mixed protein. The above result indicated that VaICE1a is possibly a ICE1 gene familys young blood.Semi-quantitative PCR results showed that VaICE1a gene indifferent tissues of Vitis a

10、murensis Rupr. was expressed degrees. under abiotic Stress, VaICE1a gene could induce expresses, The expression Level was not obvious difference,compare with control groupes.VaICE1b gene cDNA span is 1653bp, according to the nucleotide analysis that it was composed 550amino acids, named VaICE1b. The

11、 cDNA sequence and the code protein sequence showed that it includes a rich s area, the nucleus localization signaling zone (NLS), to transfer the membrane area. Homology analysis showed that the deduced VaICE1 protein was highly homologous to the Vitis vinifera (XP-002275711). Malus、Populus trichoc

12、arpa、Vitis vinifera (XP-002284528)、Arabidopsis thaliana、Triticum aestivum, the identity are 64%、68%、68%、99%、52%、73%, respectively. Sequence in C terminal is high conserved, and it has bHLH typical structure. Phylogenetic analysis also indicated that VaICE1b belongs to the ICE1 gene family. Tertiary

13、Structure analysis that it is mixed protein. The above result indicated that VaICE1a is possibly a ICE1 gene familys new member. Semi-quantitative PCR results showed that VaICE1b gene indifferent tissues of Vitis amurensis Rupr. was expressed degrees. The expression is relative highly compare with c

14、ontrol groupes.We constructed plant overexpression vector of VaICE1a of VaICE1b.【關(guān)鍵詞】山葡萄 抗寒性 VaICE1a VaICE1b 克隆 表達(dá)【英文關(guān)鍵詞】Vitis amurensis Rupr cold resistance VaICE1a VaICE1b Cloning Express【目錄】山葡萄兩個(gè)ICE1同源基因的克隆與表達(dá)特性研究摘要6-7Abstract7-8縮略詞11-12前言12-181 材料與方法18-261.1 試驗(yàn)材料181.1.1 材料181.1.2 菌株和質(zhì)粒181.1.3 酶和

15、生化試劑181.2 試驗(yàn)方法18-261.2.1 山葡萄的組培擴(kuò)繁181.2.2 山葡萄ICE1基因cDNA的克隆18-211.2.3 山葡萄ICE1基因全長的克隆21-221.2.4 山葡萄ICE1基因的表達(dá)特性研究22-231.2.5 VaICE1a和VaICE1b基因雙元表達(dá)載體的構(gòu)建及鑒定23-262 結(jié)果與分析26-512.1 山葡萄CDNA的分析26-362.1.1 山葡萄組培苗總RNA的提取262.1.2 cDNA全長的克隆與序列分析26-302.1.3 VaICE1a和VaICE1b基因多序列比對及其系統(tǒng)發(fā)育30-342.1.4 山葡萄ICE1蛋白的結(jié)構(gòu)及其功能分析34-362

16、.2 基因組結(jié)構(gòu)分析36-422.2.1 山葡萄VaICE1a、VaICE1b基因組全長的克隆36-372.2.2 VaICE1a基因組結(jié)構(gòu)分析37-402.2.3 VaICE1b基因組結(jié)構(gòu)分析40-422.3 ICE1基因在山葡萄中的半定量RT-PCR表達(dá)分析42-472.3.1 VaICE1a基因在山葡萄不同組織中的表達(dá)42-432.3.2 VaICE1b基因在山葡萄不同組織中的表達(dá)432.3.3 山葡萄ICE1基因在非生物脅迫下的表達(dá)分析43-472.4 植物表達(dá)載體的構(gòu)建47-513 討論51-533.1 ICE1基因結(jié)構(gòu)分析513.2 山葡萄ICE1基因的表達(dá)51-534 結(jié)論53-54參考文獻(xiàn)54-59致謝59several group number, then with b a, =c,c is is methyl b two vertical box between of accurate size. Per-23 measurement, such as proceeds of c values are equal and equal to the design value, then the vertical installation accurate. For example a, b, and c