摘要：

丝氨酸羟甲基转移酶(SHMT)是一种保守的磷酸吡哆醛依赖性酶,催化丝氨酸与甘氨酸间的可逆转换,同时也是核苷酸生物合成、叶酸和氨基酸代谢所必需的酶,在植物发育过程中起重要作用,亦可以提高作物抵御逆境胁迫的能力。天津农学院园艺园林学院吴颖课题组前期对西瓜在低温胁迫下进行转录组、代谢组联合分析,发现ClSHMT8在低温胁迫下表达量显著上调,推测该基因正调控西瓜的低温胁迫,深入探究西瓜ClSHMT8的生物学功能及在低温和盐胁迫中的调控机制。以西瓜自交系TN07011为材料,利用RT-PCR方法克隆ClSHMT8并进行生物信息学分析,构建过表达载体,转化拟南芥,对T₃转化株系分别采用低温和盐胁迫处理,通过表型和生理指标测定,探究ClSHMT8在非生物胁迫中的功能。结果表明:ClSHMT8全长1 749 bp,编码582个氨基酸,NCBI-CDD保守域预测显示该蛋白具典型SHMT结构域。系统进化树分析表明,该蛋白与黄瓜、苦瓜的同源蛋白的亲缘关系较近。将ClSHMT8在拟南芥中异源表达,共获得3个T₃株系,发现T₃拟南芥与野生型比较莲座叶面积增加、开花期提前、种子粒径亦显著高于野生型。在低温和盐胁迫下,T₃植株的可溶性蛋白(SP)、叶绿素(Chl)含量和过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)等抗氧化酶活性均显著高于野生型,而丙二醛(MDA)含量显著低于野生型。综上表明,ClSHMT8不仅能够正调控植株的生长发育,还可提高植株对非生物胁迫的耐受性,对拟南芥响应非生物胁迫环境具有重要作用。

关键词: 西瓜, ClSHMT8, 植物超表达载体, 非生物胁迫, 功能分析

Abstract:

Serine hydroxy methyl transferase(SHMT) is a conserved pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion between serine and glycine. It is also an essential enzyme for nucleotide biosynthesis, folate, and amino acid metabolism, playing a critical role in plant development and enhancing the ability of crops to resist stress.The watermelon inbred line TN07011 was used as the material.ClSHM78 was cloned by RT-PCR technology and subjected to bioinformatics analysis.An overexpression vector was constructed and transformed into Arabidopsis thaliana. The T₃ transformed lines were subjected to low-temperature and salt stress treatments,by determining phenotypic and physiological indices, the function of ClSHMT8 in abiotic stress was explored.The results showed that ClSHMT8 was 1 749 bp in full length, encoding 582 amino acids. NCBI-CDD conserved domain prediction indicated that the protein contained a typical SHMT domain.Phylogenetic tree analysis showed that this protein shares a close genetic relationship with its homologous proteins from cucumber and bitter melon.Heterologous expression of ClSHMT8 in Arabidopsis thaliana yielded three T₃ lines, which exhibited significantly larger rosette leaf area, earlier flowering period, and larger seed size compared to the wild type. Under low-temperature and salt stress, the T₃ plants showed significantly higher contents of soluble protein(SP) and chlorophyll(Chl), as well as activities of antioxidant enzymes such as peroxidase(POD), superoxide dismutase(SOD), and catalase(CAT), while the content of malondialdehyde(MDA) was significantly lower than that of wild type. Taken together, ClSHMT8 not only positively regulates plant growth and development, but also enhances plant tolerance to abiotic stresses.These results demonstrate that ClSHMT8 plays an important role in the response of Arabidopsis thaliana to abiotic stress environments.

Key words: Watermelon, ClSHMT8, Plant overexpression vector, Abiotic stress, Functional analysis

中图分类号: 

• S651

• Q78基因工程(遗传工程)