棉花GhSPS1的克隆及表达模式分析

Cloning and Analysis Expression Patterns of GhSPS1 Gene in Cotton

【目的】克隆棉花蔗糖磷酸合成酶基因(sucrose-phosphate synthase,SPS),并分析该基因在组织和纤维发育不同阶段及不同非生物胁迫下的表达模式。【方法】采用genome-walking及over-lap PCR方法,获得了棉花中一个蔗糖磷酸合成酶基因——GhSPS1,通过半定量PCR法检测GhSPS1在不同非生物胁迫条件下的表达模式,采用实时荧光定量PCR法对GhSPS1及GhSUS3、GhINV、GhCESA4进行组织和纤维发育特异性表达分析。【结果】克隆得到的GhSPS1全长4 545 bp,包含10个内含子、11个外显子,其开放读码框为3 108 bp,编码1 035个氨基酸,且该基因在ABA、低温处理条件下表达量增加,在高温胁迫下,表达量先升高后降低。实时荧光定量PCR结果显示,GhSPS1及GhSUS3在各种组织中都有表达,其中,在0 DAF、3 DAF的胚珠及18 DAF的纤维中表达量最高,而GhINV、GhCESA4则分别在3—15 DAF的纤维、18 DAF的纤维中表达量最高。【结论】克隆得到的GhSPS1属于SPSA基因家族,能参与ABA、低温、高温等非生物胁迫应答,且可能在纤维发育的起始期及次生壁增厚初期发挥作用。

【Objective】 The GhSPS1 gene was cloned and analyzed the expression patterns under abiotic stresses treatments.In different tissues and at fiber developmental stages,transcriptional patterns of the gene were also detected.【Method】 The DNA and cDNA sequence of GhSPS1 were cloned from cotton by genome-walking and over-lap PCR,respectively.The expression patterns of GhSPS1 were analyzed by using semi-quantitative PCR when the cotton seedlings were treated with abiotic stresses.The characteristics of four genes,GhSPS1,GhSUS3,GhINV and GhCESA4,were examined by real-time quantitative PCR at fiber developmental stages.【Result】 Comparison GhSPS of DNA and cDNA sequence,the results showed that GhSPS1 is 4 545 bp,with 3 108 bp ORF encoding 1 035 amino acids,include 10 introns and 11 extrons.The results of semi-quantitative PCR exhibited that the mRNA levels of GhSPS1 increased when the cotton seedlings were treated with ABA and low temperature.In high temperature treatment,the expression of GhSPS1 decreased firstly,and then followed with a little increase.Analysis of the genes expression patterns by real-time quantitative PCR showed that GhSPS1 and GhSUS3 expressed in all tissues,but the highest expression was in 0 DAF ovules,3 DAF and 18 DAF fibers.On the contrast,GhINV transcripts were expressed higher in 3-15 DAF fibers than in other tissues.And GhCESA4 transcripts had the highest expression level in 18 DAF fibers.【Conclusion】 GhSPS1 belongs to SPSA family,which can be induced by using ABA,low and high temperature treatments.The expression of the gene might have close relationship with initial fiber development and secondary wall thickening.