小麦CpFBA基因的多样性及其对低温处理的响应

The Diversity of Wheat CpFBA Genes and its Responsive to Low Temperature

叶绿体果糖-1,6-二磷酸醛缩酶(chloroplast fructose-1,6-biphosphate aldolase,CpFBA)是Calvin循环碳固定过程中的一个关键酶。该酶在光合作用中具有重要的功能,在一些非生物胁迫响应中也起重要的调控作用。本文用RT-PCR、RACE方法从小麦返白系中克隆到三个小麦CpFBA(TaCpFBA)的cDNA序列,预测其中两个编码388AA、另一个编码309AA;在对照矮变1号中克隆到两个TaCpFBA的cDNA序列,预测分别编码388AA和373AA。两个材料中得到的388AA序列完全一致,预测1-37位氨基酸残基是叶绿体定位导肽序列。根据cDNA全长序列设计引物,从返白系和矮变1号中分别扩增得到TaCpFBA的基因组DNA序列,两个序列均包含六个外显子。构建TaCpFBA-eGFP融合表达载体并转化拟南芥原生质体,对基因表达产物进行了亚细胞定位,证明TaCpFBA基因表达产物定位于叶绿体中。用Real TimePCR对小麦CpFBA在低温条件下的表达模式进行了初步研究,结果表明,低温条件下矮变1号中CpFBA的表达先升高后降低,而在低温敏感的小麦返白系中,CpFBA的表达先降低后逐渐升高,揭示了小麦CpFBA在不同的低温响应机制中的表达差异。

Chloroplast fructose-1,6-bisphosphate aldolase（CpFBA,aldolase） is a key enzyme involved in the carbon fixation of Calvin Cycle which is important metabolism pathway in photosynthesis.It has been reported that this enzyme participate in the response to different types of abiotic stresses in land plants.In this article,three cDNA sequences encoding wheat CpFBA（TaCpFBA） were cloned by RACE（Rapid-amplification of cDNA ends） and RT-PCR from winter wheat variety Albinism line,two of these may encode 388 amino acids,the other one encodes 309 amino acids.Another two different cDNA sequences come from the control wheat Aibian 1,which encode 388 and 373 amino acids separately.Two 388 amino acids sequences from different materials are identity.We predicted the sequence has a 37 amino acids chloroplast transit peptide by software online.According the cDNA sequences,two primers were designed to amplify TaCpFBA genomic DNA.A 2 669 bp sequence from Albinism line,and a 2 630 bp sequence from Aibian 1 were obtained,both sequences contain 6 exons.The TaCpFBA-eGFP fusion vector was constructed.After the vector was transferred into Arabidopsis protoplasts,the transit expression of TaCpFBA-eGFP fusion gene indicated that the TaCpFBA gene encode a chloroplast protein.Furthermore,the expression pattern of TaCpFBA in cold condition was investigated in albinism line and Aibian 1 by quantitative Real Time PCR.We found that,the TaCpFBA trancription of Aibian 1 rose first and then went down,but in albinism line,the wheat sensitive to low temperature,first decreased obviously,then increased gradually.The results suggested the inconsistent expression of TaCpFBA gene in different cold responsive mechanism.