桑树橡胶延伸因子基因的克隆与表达分析

Molecular Cloning and Expression Analysis of Rubber Elongation Factor Gene in Mulberry(Morus L.)

桑树乳汁在桑树的生长和防御过程中起重要作用,乳汁中的糖苷酶抑制剂还是治疗糖尿病的有效成分。通过桑树cDNA文库中的序列比对,发现一段与乳汁合成相关的基因片段,采用RACE方法获得该基因的全长序列,基因cDNA全长1 145 bp,编码区759 bp,编码252个氨基酸残基,将该基因命名为桑树橡胶延伸因子基因(GenBank登录号:GQ466080)。为探究该基因的功能,通过RT-PCR的方法分析该基因在桑树不同组织器官的表达,结果表明该基因在桑树幼叶中的表达量最高,其次是茎和芽,在根中的表达量最低。对桑树叶片进行细胞分裂素处理,发现细胞分裂素处理后该基因表达量有减少的趋势,在细胞分裂素处理的叶片中,正在伸展的幼叶中该基因的表达量要高于刚出现的幼叶和成熟叶。与桑树中已知的其它功能基因相比,桑树橡胶延伸因子在桑树正常生长中的表达量远高于桑树Na+/H+逆向转运蛋白基因MaNHX和桑树抗冻蛋白基因WAP27的表达量。从以上结果初步推测桑树橡胶延伸因子基因在桑树的生长发育中起重要作用。

Mulberry latex plays an important role in mulberry growth and defense.In addition,the glycosidase inhibitor in it is an active ingredient of antidiabetes.In a mulberry cDNA library,a gene fragment which is related to latex synthesis was identified through sequence alignment.Its full-length cDNA sequence was obtained by means of RACE and was designated as mulberry rubber elongation factor gene（MaREF,GenBank accession number GQ466080）.It is 1 145 bp long and has an open reading frame of 759 bp which encodes 252 amino acid residues.Expression analyses of MaREF gene in various mulberry tissues and organs through RT-PCR indicated that it had the highest expression level in young leaves,the moderate levels in stems and buds,and the lowest level in root.Treatment of mulberry leaves with cytokinin could reduce the expression level of MaREF gene.Among the cytokinin treated leaves,the expression level in young leaves at the expanding stage was higher than the newly emerged young leaves and mature leaves.Compared with other functional genes in mulberry,the expression level of MaREF gene was far much higher than the levels of mulberry Na^＋/H^＋ antiporter gene MaNHX and mulberry cold acclimation gene WAP27.The above results preliminarily suggest that MaREF gene plays an important role in mulberry growth and development.