山药异淀粉酶基因克隆及其在淀粉代谢中的作用

Cloning of Isoamylase Gene in Yam and Its Role in Starch Metabolism

为了明确异淀粉酶基因(ISA 3)在山药淀粉代谢中的作用,该研究以‘毕克齐’和‘大和长芋’山药为试验材料,测定了块茎中淀粉及组分含量和异淀粉酶活性等;采用RT-PCR技术克隆了ISA 3,并进行生物学分析及山药块茎不同膨大期和不同组织间ISA 3基因的表达等。结果表明:(1)山药‘大和长芋’的直链淀粉、支链淀粉和总淀粉含量均显著高于‘毕克齐’,且两品种的淀粉含量随生长发育的变化均呈先升高后降低的趋势,并均于种植后120 d时达到最高,但‘毕克齐’的异淀粉酶(ISA)活性在整个膨大期均高于‘大和长芋’。(2)成功克隆获得山药ISA 3开放阅读框长1584 bp,编码527个氨基酸;ISA3为亲水性蛋白。(3)不同品种块茎在膨大时期的ISA 3基因表达趋势不同,‘毕克齐’中呈先显著上调随后下调,而在‘大和长芋’中表达总体下调,且在山药的叶、茎和块茎中均有表达,存在明显的组织特异性。(4)ISA活性与山药淀粉及支链淀粉含量呈显著和极显著正相关关系,但ISA活性与ISA 3的表达量呈负相关关系。研究表明,异淀粉酶参与了山药块茎中淀粉的合成,且主要对支链淀粉的合成起关键作用,ISA 3基因的表达可能对异淀粉酶活性和淀粉的合成起负调控作用。

In order to study the role of the isoamylase gene(ISA 3)in the metabolism of yam tuber starch,we used Bikeqi yam and Dahechangyu yam as the measure to determine the starch and its component content and isoamylase(ISA)activity.The RT-PCR technology has been used to clone the open reading frame of ISA 3.The bioinformatics analysis were performed.Expression of ISA 3 gene in different swelling stages tuber and among different tissues of yam was analyzed.The results showed that:(1)the amylose,amylopectin and total starch contents of Dahechangyu yam are significantly higher than Bikeqi yam,and the starch content of the two varieties showed a trend of first increase and then decrease with the change of growth and development,it reached the highest level at 120 d after planting.However,the ISA activity of Bikeqi yam was higher than that of Dahechangyu yam during the whole expansion period.(2)We successfully isolated an isoamylase gene(ISA 3)from yam variety Bikeqi and Dahechangyu.Its sequence length is 1584 bp and encodes 527 amino acids.ISA3 is a hydrophilic protein and non-transmembrane structure.(3)In different periods of yam tuber expansion of 2 varieties with different starch contents,ISA 3 gene expression increases first and then decreases in Bikeqi yam,but gradually decreases in Dahechangyu yam.It is expressed in the leaves,stem and tuber,and there is obvious tissue specificity.(4)ISA activity was extremely significantly and significantly positively correlated with yam starch content and amylose content.Nevertheless,ISA activity was negatively correlated with the expression of ISA 3.This result suggests that ISA is involved in the synthesis of starch in tubers and play a key role in the synthesis of amylose,and ISA 3 expression may play negative regulation of ISA activity and starch synthesis.