棉花腺苷高半胱氨酸水解酶cDNA的克隆、表达及染色体定位

Cloning, Expression, and Mapping of S-adenosyl-L-homocysteine Hydrolase (GhSAHH) cDNA in Cotton

腺苷高半胱氨酸水解酶是调节细胞内甲基反应的一个关键酶。通过对高品质纤维陆地棉品系7235的棉纤维混合cDNA文库随机测序,得到一个棉花腺苷高半胱氨酸水解酶(编号:g073a03a,GhSAHH)的cDNA序列。该cDNA序列长1598bp,利用5′RACE技术得到上游318bp的片段,序列拼接获得全长为1916bp的cDNA序列,ORF为1458bp,编码485个氨基酸,其理论上的等电点pI=5.69,分子量MW=53.2kD。该基因在不同组织、器官中均表达,在根、下胚轴和纤维发育早期优势表达。根据Southern杂交结果推测GhSAHH基因在陆地棉基因组中为单拷贝。利用本实验室陆地棉遗传标准系TM-1和海岛棉海7124培育的含140个单株的BC1作图群体,将GhSAHH基因定位在第20号染色体上。

S-adenosyl-L-homocysteine hydrolase （SAHH） is a key enzyme in the regulation of intraeellular methylation reactions. Though the SAHH function has already been elucidated in various living creature bodies, it is still not explicit in growth and development of cotton fiber. Therefore, research of SAHH may provide effectively basis to clarify the mechanism of cotton growth and development. In this paper, we obtained a full length cDNA fragment of SAHH in cotton and further finished its expression, mapping and Southern blotting analyses. A cDNA clone encoding S-adenosyl-homocysteine hydrolase （Library No. g073a03a, GhSAHH） was isolated from cotton fiber cDNA library of 7235 germplasm line with elite fiber quality in Gossypium hirsutum L. The length of this cDNA clone was 1 598 bp. Further, a 318 bp-long-upstream fragment was obtained via 5RACE technique. The full length of the cDNA clone was 1 916 bp with its open reading frame encoded 485 amino acids. The putative protein of this gene had an isoelectric point of 5.69 and a calculated molecular weight of 53.2 kD. Judging from its expression characters, GhSAHH is expressed in cotton cells, especially in root, hypocotyl and at early stage of fiber developmental period. Southern blotting analyses showed there was only one copy of GhSAHH in the genome of upland cotton. Using the BC1 mapping population with 140 individuals derived from the hybridization between the upland cotton standard line TM-1 and the sea-island cotton cultivar Hai7124, and TM-1 as recurrent parent, GhSAHH was localized on the chromosome 20. We have already constructed plant express vector and RNAi vector of GhSAHH, the transgenic research on cotton is undertaking.