青杄分子伴侣PwTCP-1基因的克隆及表达分析

Cloning and expression analysis of PwTCP-1 in Picea wilsonii

分子伴侣TCP-1在调节植物生长发育过程中具有重要作用,但在林木生长发育及对非生物逆境胁迫响应机制方面的研究较少。从青杄c DNA文库中筛选出1个分子伴侣PwTCP-1(T-complex polypeptide 1)片段,通过RACE-PCR技术获得PwTCP-1的末端序列,经过与EST序列拼接,获得PwTCP-1c DNA全长序列。使用clustalx、Mega5.0、DNAMAN和Expasy等工具对PwTCP-1的理化性质、二级结构和三级结构进行生物信息学分析。结果显示,PwTCP-1c DNA全长为2024 bp,开放阅读框1 605 bp,编码534个氨基酸残基的蛋白质,其理论分子质量为59.2k Da,理论等电点为6.12,富含α-螺旋。通过RT-q PCR技术检测了PwTCP-1组织特异性表达、激素响应模式及在种子萌发过程中表达量变化。结果表明:PwTCP-1在青杄各个组织中均有表达,在花粉中表达量最高,在茎中表达量最低;PwTCP-1表达量随着种子萌发先上升,在第4天达到最高,之后缓慢下降。PwTCP-1能响应多种激素处理。油菜素内酯(brassinolide,BR)和赤霉素(gibberellin,GA)分别处理后,PwTCP-1的表达量均显著降低,而茉莉酸甲酯(methyl jasmonate,Me JA)处理后该基因表达量极显著升高。这些结果显示,PwTCP-1可能参与植物种子萌发过程并响应了多种激素反应过程。

Molecular chaperonins belong to a conserved protein family and play crucial roles in protein folding and assembling. The molecular chaperonin TCP-1 has been shown to be involved in the regulation of plant development. However, little is known of its molecular mechanism in the development of woody plants and their response to abiotic stresses. The PwTCP-1 （T-complex polypeptide 1 ） gene, encoding molecular chaperonin, was isolated from the cDNA library of Picea wilsonii. The full-length cDNA of PwTCP-1 was obtained by jointing an EST fragment and the end sequence of PwTCP-1 with RACE-PCR. Several bioinformatic tools, such as clustalx, Mega5.0, DNAMAN and Expasy, were used to analyze its physical and chemical properties, as well as secondary and tertiary structures of PwTCP-1. Bioinformatic analysis showed that PwTCP-1 cDNA had a full length of 2 024 bp and a major open reading frame of 1 605 bp, encoding a protein of 534 amino acids, with a molecular mass of 59.2 kDa and a theoretical isoelectric point of 6.12. PwTCP-1 is rich in a-Helix and highly conservative. The expression patterns of PwTCP-1 in various tissues, phytohormone responses and seed germination were investigated by RT- qPCR. The results of tissue-specific expression revealed that it was expressed in every tissue of P. wilsonii, with the expression level in pollen the highest among all tissues and the lowest in the stem. During seed germination, the expression level of PwTCP-1 increased, peaked at the fourth day and then decreased slowly. It should be noted that PwTCP-1 can respond to several phytohormone treatments. The expression level of PwTCP-1 was significantly inhibited by both brassinolide and gibberellin treatments, but up-regulated by a methyl jasmonate treatment. These results suggest that PwTCP-1 is involved in the process of seed germination and a response to several phytohormones.