摘要
扩张蛋白(expansin,EXP)能使植物细胞壁松驰,在果实成熟软化过程中起重要作用。克隆柿果实扩张蛋白基因,对研究柿果实成熟软化机理,进而有效控制柿果实成熟软化进程,延长保鲜期具有重要意义。
本研究以中国柿(Diospyros Kaki L.cv Fuping jianshi)为试材,在果实不同发育时期分别提取总RNA,进行RT-PCR结合RACE技术扩增得到五个扩张蛋白基因cDNA全长序列,并构建了原核表达载体。取得的主要结果如下:
1.柿果实扩张蛋白基因的cDNA克隆以柿果实不同发育时期提取的总RNA为模板,利用RT-PCR结合RACE技术扩增得到五个扩张蛋白基因:成熟期CDK-Exp3(1168bp),转色期CDK-Exp4(1120bp)和CDK-Exp5(1018bp),膨大期CDK-Exp6(1129bp)和CDK-Exp7(1121bp);其中CDK-Exp3是包含完整5’和3’末端的全长cDNA序列,CDK-Exp4、CDK-Exp5、CDK-Exp6和CDK-Exp7是包含完整ORF和3’末端的cDNA序列。
2.柿果实扩张蛋白基因的生物信息学分析柿果实扩张蛋白基因的编码区ORF为765bp,编码254个氨基酸残基,具有扩张蛋白的保守序列(如HFD基元)和不变残基,即N端有8个半胱氨酸残基,C端有4个色氨酸残基;预测分子量27150.36 D,等电点7.587。CDK-Exp3编码的蛋白前27个氨基酸是典型的信号肽结构。由柿果实CDK-Exp3推导的氨基酸序列与CDK- Exp4、CDK- Exp5、CDK- Exp6、CDK- Exp7的同源性分别为99.2%、99.2%、98.4%、98.8%,与CDK- Exp1、CDK- Exp2的同源性在80%左右;与杏、樱桃、梨、苹果的同源性在80%以上,与桃、李、草莓、黄瓜的同源性在70%以上,与番茄的同源性为65.7%;同在转色期克隆到的CDK- Exp4与CDK- Exp5的氨基酸序列同源性为100%,但二者的核苷酸序列在3’端非编码区相差102bp。
3.柿果实扩张蛋白基因的原核表达设计带有酶切位点的引物,扩增出去掉信号肽结构的CDK-Exp3的ORF序列,经BamHⅠ和XhoⅠ双酶切,定向克隆到表达载体pET-32(a+)中,获得柿果实扩张蛋白基因原核表达载体pET- CDK-Exp3。将构建的表达载体pET-CDK-Exp3转化大肠杆菌BL21(DE3),诱导表达得到大小约为44kDa的蛋白;诱导表达的最佳条件为:0.1mM IPTG,37℃诱导表达6h。
Expansin can cause plant cell wall loosening, and plays an important role in fruit ripening and softening. Therefore, cloning expansin gene from persimmon fruit is very important for its ripening and softening research, and for further measures to prolong storage period.
Persimmon (Diospyros kaki L.cv.Fuping Jianshi ) fruit was used as material in this research. The total RNA from different stages of persimmon fruit were extracted, five full length expansin cDNA were obtianed by RT-PCR and RACE, and prokaryotic expression vector pET-CDK-Exp3 was constructed. The results are as follows.
1. Cloning of expansin cDNA from persimmon fruit
Using the total RNA from different stages of persimmon fruit as the template, five expansin cDNA: CDK-Exp3(1168bp)from mature stage, CDK-Exp4(1120bp)and CDK-Exp5(1018bp)from colour-changed stage, CDK-Exp6(1129bp)and CDK-Exp7(1121bp)from fruit expanding stage were isolated by RT-PCR and RACE. CDK-Exp3 was the full length cDNA containing 3’and 5’end sequence. The other four contained the complete ORF and 3’end sequence.
. Bioinformatics analysis of persimmon fruit expansin gene
The open reading frame of persimmon fruit expansin gene was 765bp and encoded 254 amino acid residues, which had the conserved sequences (such as HFD) and invariant residues (8 cysteine residues in N-terminal and 4 tryptophan residues in C-terminal)of expansin. Its predict molecular weight was 27150.36 Da, and isoelectric point was 7.587. The protein encoded by CDK-Exp3 had the structure of signal peptide composed of the first 27 amino acid residues.
The homology of deduced amino acid sequence of CDK-Exp3 with CDK-Exp4, CDK-Exp5, CDK-Exp6 and CDK-Exp7 were respectively 99.2%, 99.2%, 98.4%and 98.8%; with CDK-Exp1 and CDK-Exp2 were about 80%; with apricot, cherry, pear and apple were more than 80%; with peach, plum, strawberry and cucumber were above 70%; with tomato was 65.7%. The homology of deduced amino acid sequence of CDK-Exp4 with CDK-Exp5, both cloned from colour-changed stage of persimmon fruit, was 100%; but the nucleotides of them had 102bp difference in 3’noncoding region.
3. Prokaryotic expression of persimmon fruit expansin gene
The ORF sequence without signal peptide of CDK-Exp3 was obtained and restricted with XhoⅠand BamHⅠ, and then inserted into pET-32(a+). pET-CDK-Exp3 expression vector was obtained after sequencing. pET-CDK-Exp3 recombinant was transferred to an E.coli strain BL21(DE3). After IPTG induction, a 44kDa fusion protein was expressed. The optimum inducement condition is: 0.1mM IPTG 37℃, 6h.
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