亚麻生氰糖苷合成关键酶CYP79基因家族的鉴定及表达分析

Identification and expression analysis of CYP79 gene family,a key enzyme for cyanogenic glycoside synthesis in flax

CYP79蛋白是生氰糖苷合成关键酶,但关于亚麻CYP79基因的研究鲜有报道。本研究对包括亚麻在内的9种作物的CYP79基因家族进行了鉴定,并分析了亚麻CYP79基因的序列特征、复制事件、共线性关系、系统进化、顺式作用元件及表达模式。结果表明,在亚麻、白亚麻、毛果杨、木薯、芝麻、高粱、大豆、葡萄及水稻中分别鉴定到9、9、3、2、5、7、6、16和4个CYP79家族成员;系统进化分析显示,CYP79基因的进化具有物种特异性;LuCYP79不均匀分布在4条染色体上,具有1~3个外显子,其启动子区含大量激素与逆境响应相关元件;共克隆到8个亚麻CYP79基因的全长DNA序列及5个成员的全长cDNA序列;LuCYP79蛋白序列长度为282~565aa,等电点为5.84~9.14,分子量为31.56~62.86kD,均为亲水性蛋白,定位于内质网;共有5对基因发生复制事件,占全部基因的77.8%,全部经历了强烈的纯化选择,其中LuCYP79-1和LuCYP79-9在拟南芥和木薯中均具有同源基因。表达分析表明,LuCYP79家族成员具有组织特异性,且各成员在不同遗传背景下表达模式不同,其中LuCYP79-1、LuCYP79-7、LuCYP79-8和LuCYP79-9在4个品种中的表达差异显著。相关分析表明,50d时的LuCYP79-1/LuCYP79-7与成熟亚麻籽中生氰糖苷含量呈极显著正相关,20d时的LuCYP79-7/LuCYP79-8及(LuCYP79-7+LuCYP79-9)/LuCYP79-8分别与成熟亚麻籽中生氰糖苷含量呈极显著正相关,初步推测其可能是亚麻籽生氰糖苷合成的关键基因。研究结果对进一步阐明亚麻CYP79蛋白的功能具有积极意义,并为培育低生氰糖苷亚麻品种提供了理论参考。

CYP79 is a key enzyme in the synthesis of cyanogenic glycoside.However,there is no systematical study of CYP79 genes in flax.In this study,we identified CYP79 gene family in 9 crops including flax,and focused on the sequence characteristics,duplication events,collinearity,evolution,cis-acting elements and expression patterns of Lu CYP79 genes.The results revealed that a total of 9,9,3,2,5,7,6,16,and 4 CYP79 family members were identified in flax,pale flax,poplar,cassava,sesame,sorghum,soybean,grape,and rice,respectively.Phylogenetic analysis showed that the evolution of CYP79 genes was species-specific.Lu CYP79 were distributed unevenly on four chromosomes and had 1–3 exons.The promoter region of Lu CYP79 contained lots of elements involved in response to hormone and stress.8 full-length DNA sequences and 5 full-length cDNA sequences of flax Lu CYP79 genes were cloned.LuCYP79 proteins contained 282–565 amino acid residues with molecular weight of 31.56–62.86 kD,and isoelectric point of 5.84–9.14.All LuCYP79 members were hydrophilic protein and located in endoplasmic reticulum.There were five pairs of Lu CYP79 genes with duplication events,accounting for 77.8%of all genes,and all the duplication genes underwent strong purification selection.Both Lu CYP79-1 and Lu CYP79-9 had homologous genes in Arabidopsis and cassava.The relative expression levels showed that LuCYP79 family members were tissue-specific and had different expression patterns under different genetic backgrounds.There were significantly different in the relative expression of Lu CYP79-1,LuCYP79-7,LuCYP79-8,and LuCYP79-9 in the four cultivars.Moreover,correlation analysis showed that Lu CYP79-1/Lu CYP79-7 in 50 days flaxseed was significantly positively correlated with the concentration of cyanogenic glycosides in mature flaxseed.LuCYP79-7/LuCYP79-8 and(Lu CYP79-7+LuCYP79-9)/LuCYP79-8 in 20 days flaxseed were significantly positively correlated with the concentration of cyanogenic glycosides in mature flaxseed,respectively.It was preliminarily speculated that they might be the key genes in the synthesis of cyanogenic glycoside in flaxseed.These results have positive significance for further elucidating the function of CYP79 proteins in flax and provide theoretical references for breeding flax varieties with low cyanogenic glycoside.