菊花花瓣伸长相关基因CmXTHs的克隆与功能验证

Cloning and Functional Verification of Chrysanthemum CmXTHs Genes Related to Petal Elongation

从杭白菊(Chrysanthemum morifolium‘Hangbaiju’)中克隆了4个细胞壁松弛和伸展相关的木葡聚糖内转糖苷酶/水解酶基因,分别命名为CmXTH1、CmXTH2、CmXTH3和CmXTH4,序列分析表明其编码的氨基酸序列N端都含有23~30个氨基酸组成的信号肽区域和保守的催化活性位点DE(I/L)DEFLG以及紧邻的N(R/A)T组成的N端糖基化位点,CmXTH1/2/3的C端均含有4个保守的半胱氨酸残基,CmXTH1/2/3/4蛋白的相似度为66.2%。系统进化分析结果表明,CmXTH1/2/3属于XTH家族Ⅰ组,CmXTH4属于Ⅱ组。实时荧光定量结果表明:(1)菊花CmXTH1/2/3/4在根、茎、叶和花蕾中均有表达。CmXTH1在根中表达量较高,CmXTH2/3在茎中表达量较高,CmXTH4在花蕾中表达量最高。(2)CmXTH1/2/3/4在花序不同部位表达量有差异,CmXTH1/4在舌状花中表达量最高,CmXTH2/3在筒状花中表达量较高。(3)CmXTH1/2/3/4在舌状花不同发育阶段表达量差异显著,CmXTH1/2在盛花期表达量最高,CmXTH3在衰老期表达量最高,CmXTH4在初开期表达量最高。病毒诱导基因沉默结果表明,CmXTH1/2/3/4沉默系的花序直径和舌状花长度与对照相比均有所减小,其中CmXTH4沉默系减小最大,分别减小了25.67%和10.42%,舌状花花瓣表皮细胞与对照相比明显变小,影响了舌状花花瓣的伸长;CmXTH2沉默系的筒状花雄蕊长度和CmXTH4沉默系的花萼直径分别与对照相比显著减小。这表明菊花CmXTH1/2/3/4基因参与了花序的开放,促进了舌状花花瓣的伸长,对于菊花的花序增大具有重要作用。

Four xyloglucan endotransglucosylase/hydrolase genes that related to cell wall loosen were cloned from chrysanthemum and named as CmXTH1,CmXTH2,CmXTH3 and CmXTH4. Sequences analysis showed that the N-terminal regions of deduced amino acid sequences all had signal peptides composed of 20–30 amino acids,conserved catalytic sites of DE（I/L）DEFLG and N-glycosylation sites composed of N（R/A）T,CmXTH1/2/3 had four cysteine in C-terminal regions,the identity of 4 CmXTH1/2/3/4 was 66.2%. The phylogenetic analysis result suggested that CmXTH1/2/3 belongs to groupⅠof XTH family,CmXTH4 belongs to groupⅡ. The real-time PCR results showed that：（1）CmXTH1/2/3/4 all had expression in root,stem and leaf. CmXTH1 was highly expressed in chrysanthemum root. CmXTH2/3 was expressed more in stem. CmXTH4 was highly expressed in alabastrum.（2）CmXTH1/2/3/4 showed different expression levels in different parts of inflorescence. CmXTH1/4 was expressed more in ray florets. CmXTH2/3 was expressed more in tubular florets.（3）CmXTH1/2/3/4 showed different expression levels in different stages of ray florets development. CmXTH1/2 was highly expressed in ray florets at bloom stage,CmXTH3 was highly expressed at senescent stage,while CmXTH4 was expressed highest at initiation stage. The virus induced gene silence results showed that,the inflorescences diameters and ray florets lengths of CmXTH1/2/3/4-silenced groups all showed decrease compared with control,the CmXTH4-silenced group decreased most by 25.67% and 10.42% respectively,the sizes of petal epidermal cells decreased obviously,influenced the extension of ray floret petal. The stamen length of CmXTH2-silenced tubular florets and the diameter of CmXTH4-silenced sepal all showed significant decrease compared with control. These results indicated that CmXTH1/2/3/4 participated in the opening of inflorescences,promoted the extension of ray floret petal, and they were important for the enlargement of chrysanthemum inflorescences.