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目的发掘陆地棉Dirigent-like蛋白基因家族,明确不同黄萎病抗性品种间该基因家族成员的多态性及表达差异。方法利用Illumina Hiseq 2000测序平台,通过RNA-Seq测序技术,获得黄萎病菌侵染陆地棉抗病品种和感病品种的Dirigent-like蛋白基因家族成员Unigene序列;采用生物信息学软件对其进行核苷酸序列及编码蛋白的多重比对和系统进化分析;针对该家族成员设计特异的quantitative real-time PCR引物,在Applied Biosystems 7500 Real Time PCR System上使用SYBR green PCR试剂盒标记反应产物,棉花Gh ACTIN为内标,采用2-ΔΔCt法分析抗病品种冀79、感病品种TM-1中该基因家族成员在0 h及受侵染1、8、24、48、72和96 h后的表达差异。结果获得12个陆地棉Dirigent-like蛋白基因家族成员;该家族成员分属3个进化群,基因间存在4个保守结构域。其同源基因在D5基因组染色体上,主要集中于第2、3、6、7、9、10、13染色体。陆地棉受黄萎病侵染后,Dirigent-like蛋白基因抗病、感病品种间差异表达,抗病品种冀79受黄萎病菌侵染1 h时上调2倍以上的有Gh DIR4、Gh DIR6、Gh DIR7、Gh DIR9、Gh DIR10和Gh DIR11,其中,Gh DIR9上调6.5倍;侵染8 h时Gh DIR1、Gh DIR4、Gh DIR7和Gh DIR11上调,有2个上调2倍以上;侵染24 h时仅Gh DIR4上调2倍以上;侵染48 h时Gh DIR1、Gh DIR4、Gh DIR7和Gh DIR11上调达到2倍以上;侵染72 h时仅Gh DIR9上调2倍以上,达到6.4倍;侵染96 h时仅Gh DIR4上调2倍以上。感病品种TM-1受侵染1 h时仅Gh DIR7上调2倍以上;受侵染8 h时Gh DIR7上调325倍,Gh DIR10上调2倍以上;侵染24和48 h时Gh DIR4、Gh DIR5、Gh DIR6、Gh DIR7、Gh DIR9、Gh DIR10急剧上调,Gh DIR7、Gh DIR9、Gh DIR10上调10倍以上;受侵染72 h时Gh DIR4和Gh DIR7还保持较高的表达量;侵染96 h时Gh DIR5、Gh DIR6、Gh DIR7、Gh DIR9、Gh DIR10和Gh DIR11保持较高的表达量。由此看出,受黄萎病侵染时抗病种质冀79 Dirigent-like蛋白基因家族成员上调表达要早于感病品种,但是,该基因家族成员在感病品种受侵染8 h后上调倍数远远高于抗病品种,而且到96 h时其表达量还较高;抗病品种上调的Dirigent-like蛋白家族成员Gh DIR4、Gh DIR7、Gh DIR9和Gh DIR11值得关注;Gh DIR6、Gh DIR7、Gh DIR9和Gh DIR10在感病品种TM-1中上调显著。此外,抗病、感病品种间Gh DIR1、Gh DIR3、Gh DIR5、Gh DIR8、Gh DIR10和Gh DIR12存在核苷酸多态性。结论 Dirigent-like蛋白与黄萎病菌侵入后棉花植株的抗性反应相关。
Objective To explore the Dirigent-like protein gene family of upland cotton (Gossypium hirsutum L.), and to determine the polymorphism and expression differences of this gene family among different Verticillium wilt resistance cultivars. Methods The Unigene sequence of Dirigent-like protein gene family of resistant and susceptible cultivars of Upland cotton to Verticillium dahliae was obtained by using Illumina Hiseq 2000 sequencing platform and RNA-Seq sequencing technology. Bioinformatics software was used to perform nuclear Nucleotide sequence and coding protein multiple alignment and phylogenetic analysis. To design specific quantitative real-time PCR primers for this family member, the reaction products were labeled with SYBR green PCR kit on Applied Biosystems 7500 Real Time PCR System. Cotton Gh ACTIN was used as an internal standard to analyze the expression of the resistant gene JI 79 using 2-ΔΔCt method. The expression of TMTIN gene family members at 0 h, 1 h, 2 h, 2 h, 24 h, 48 h, 72 h and 96 h after infection difference. As a result, 12 members of Dirigent-like protein gene family from upland cotton were obtained. The members belong to 3 evolutionary groups and there are 4 conserved domains among them. The homologous genes in the D5 genome chromosome, mainly in the first 2,3,6,7,9,10,13 chromosome. After inoculation by Verticillium wilt, the Dirigent-like protein genes were resistant to the disease and the susceptible varieties were differentially expressed. The resistance of the resistant cultivar Hebei 79 to Drosophila melanogaster increased more than twice as much as that of Gh DIR4 and Gh DIR6 , Gh DIR7, Gh DIR9, Gh DIR10 and Gh DIR11, among which, Gh DIR9 was up-regulated by 6.5 times. Upstream Gh DIR1, Gh DIR4, Gh DIR7 and Gh DIR11 up-regulated by 2 h Gh DIR4 up-regulated more than 2-fold when h and 48h respectively; up-regulated more than 2 times Gh DIR1, Gh DIR4, Gh DIR7 and Gh DIR11 when infected for 48 h; Gh DIR4 up-regulated more than 2-fold at 96 h. Gh DIR7 was only upregulated by more than 2 folds at 1 h infected with susceptible cultivar TM-1, 325 fold by Gh DIR7 at 8 h and up-regulated by more than 2 folds of Gh DIR10 at 8 h after infection; Gh DIR4, Gh at 24 and 48 h DIR5, Gh DIR6, Gh DIR7, Gh DIR9 and Gh DIR10 were up-regulated dramatically. Gh DIR7, Gh DIR9 and Gh DIR10 were up-regulated more than 10 folds. Gh DIR4 and Gh DIR7 maintained high expression level at 72 h Gh DIR5, Gh DIR6, Gh DIR7, Gh DIR9, Gh DIR10 and Gh DIR11 maintained high expression levels at 96 h. It can be seen from the above that the members of resistant gene 79 Jiri-like protein gene family were up-regulated earlier than the susceptible ones when they were infected by Verticillium wilt. However, The up-regulation was much higher than that of the resistant varieties, and its expression level was still high at 96 h. The members of the Dirigent-like protein family Gh DIR4, Gh DIR7, Gh DIR9 and Gh DIR11 which were up-regulated by the resistant varieties were worthy of attention. Gh DIR6, Gh DIR7, Gh DIR9 and Gh DIR10 were significantly up-regulated in susceptible cultivar TM-1. In addition, there were nucleotide polymorphisms in Gh DIR1, Gh DIR3, Gh DIR5, Gh DIR8, Gh DIR10 and Gh DIR12 between resistant and susceptible cultivars. Conclusion Dirigent-like protein is related to the resistance of cotton plants to invasion of Verticillium dahliae.