引用本文:陈蒙, 刘海峰.山葡萄C4H基因的克隆表达及遗传转化分析[J].西南大学学报(自然科学版),2019,41(10):11~21
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山葡萄C4H基因的克隆表达及遗传转化分析
陈蒙, 刘海峰
延边大学 农学院, 吉林 延吉 133002
摘要:
通过生物学技术来研究C4H基因在山葡萄着色过程中的作用,进而揭示山葡萄果皮着色的分子机理;利用RT-PCR技术克隆了山葡萄C4H基因的全长cDNA序列,并对该蛋白进行生物信息学分析,预测其功能;利用实时荧光定量PCR检测C4H基因在山葡萄8个不同转色时期的表达量,将克隆获得的山葡萄C4H基因完整的ORF连接到原核表达载体pET28a上,转化到大肠杆菌E.coli BL21(DE3),并通过不同浓度的IPTG诱导表达,SDS-PAGE检测表达产物.为了验证山葡萄C4H基因的功能,构建了表达载体pC C4H并转化农杆菌GV3101.用菌液浸泡花序法对拟南芥进行遗传转化,在含50 mg/L Kan的培养基上对T0代种子进行筛选.克隆获得的山葡萄C4H cDNA全长1 735 bp,开放阅读框1 518 bp,编码505个氨基酸,该基因表达产物分子质量为57.70 KDa,等电点值9.06.C4H基因在山葡萄果皮转色各个时期均存在表达;该基因原核表达产物与预期大小一致,表明原核表达成功,拟南芥遗传转化先后得到3个阳性幼苗.对移栽成活的2株抗性植株进行PCR检测为阳性,2株叶片颜色均变成紫红色;经花色素苷质量浓度的测定表明其质量浓度比对照组植株高出3倍.在拟南芥中花色素苷质量浓度虽然较低,但还是能少量合成,说明其花色素苷生物合成途径是开通的,只是积累的量较少.
关键词:  山葡萄  克隆  C4H基因  原核表达  遗传转化
DOI:10.13718/j.cnki.xdzk.2019.10.002
分类号:Q75
基金项目:国家自然科学基金项目(31260067);吉林省教育厅“十三五”科学技术研究规划项目(吉教科合字[2016]第255号).
Cloning Expression and Genetic Transformation Analysis of Vitis amurensis C4H Gene
CHEN Meng, LIU Hai-feng
Agricultural College of Yanbian University, Yanji Jilin 133002, China
Abstract:
[Objective] To study the effect of C4H gene in the staining process of Vitis amurensis via biological technology, thus revealing the molecular mechanism of staining on skin of this fruit.[Methods] Reverse transcription polymerase chain reaction (RT-PCT) technology was used to clone the full length cDNA sequence of V. amurensis C4H gene and bioinformatic analysis was carried out of this protein to predict its function. Real-time fluorescence quantitative PCR was used to detect the expression level of C4H gene in 8 different color transition periods of V. amurensis and its complete ORF obtained via cloning was connected to the prokaryotic expression vector pET28a and transformed to E. coli BL21 (DE3). The expression was induced by IPTG of different concentrations and the expression product was detected by SDS-PAGE. In order to verify the function of C4H, the expression vector pCC4H was established and Agrobacterium GV3101 was transformed. Genetic transformation was carried out on Arabidopsis by soaking the inflorescences in liquid bacteria. T0 generation seeds were screened out on the culture medium containing 50 mg/L Kan.[Results] The full length of V. amurensis C4H cDNA obtained via cloning was 1, 735 bp, and the open reading frame was 1, 518 bp, encoding 505 amino acids. The molecular volume (MV) of this gene expression product was 57.70 KDa and the value of isoelectric point was 9.06. C4H gene was expressed in every stage of skin color transformation of V. amurensis. The prokaryotic expression product of the gene was consistent with the expected size, indicating that the prokaryotic expression was successful. Three positive seedlings were obtained from the genetic transformation of Arabidopsis. PCR detection of the two resistant plants which survived after transplanting was positive and the leaves of both plants turned purple. In determination of anthocyanin, its content was 3 times higher than that of plants in the control group.[Conclusion] Although the content of anthocyanin in Arabidopsis is on a relatively low level, it still can be synthesized in a small amount, indicating that the biosynthetic pathway of its anthocyanin is open, though with a small accumulation amount.
Key words:  Vitis amurensis  cloning  C4H  prokaryotic expression  genetic transformation
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