苦荞WOX家族全基因组鉴定及响应愈伤诱导率表达分析

doi:10.3864/j.issn.0578-1752.2021.17.002

摘要/Abstract

摘要：

【目的】全基因组鉴定苦荞WOX（WUSCHEL-related homeobox）基因,揭示其基因家族成员序列特征、基因表达模式及与出愈率的相关性,为突破苦荞再生及遗传转化难题提供理论基础。【方法】基于同源性搜索策略,以拟南芥WOX基因蛋白为参考序列,进行苦荞全基因组比对,获得苦荞WOX基因家族成员蛋白及核酸序列。基于蛋白同源性及保守结构域分析,鉴定出苦荞WOX基因家族所有成员。同时使用TBtools软件展示FtWOXs家族成员基因结构、保守结构域及启动子顺式作用元件特征。比较分析WOX基因家族成员在苦荞与拟南芥之间的基因组共线性。基于邻近法,利用MEGA X软件构建苦荞、拟南芥和水稻WOX基因家族成员蛋白序列系统进化树。以MS+2,4-D 3.0 mg·L^-1+6-BA 1.0 mg·L^-1为愈伤诱导培养基,下胚轴为外植体,选取70份苦荞品种诱导愈伤组织,评价不同基因型的出愈率。qRT-PCR比较分析高、低出愈率苦荞品种间FtWOXs基因表达水平。基于Pearson相关系数分析出愈率与FtWOXs基因家族成员表达相关性。【结果】共鉴定出30个苦荞WOX基因成员,在苦荞8条染色体上呈现不均匀分布。系统进化树表明30个苦荞WOX基因可划分为3大类,不同类群中WOX基因包含不同的保守结构域,主要的保守结构域为HD（Homeodomain）、START和MEKHLA结构域。保守基序分析表明,FtWOXs基因家族成员所含保守基序数目的范围为2—10个。基因结构分析表明,FtWOXs基因家族成员所含外显子数目的范围为2—18个。顺式作用元件分析表明FtWOXs基因启动子富含26个不同种类的顺式作用元件。系统进化分析表明,30个苦荞、15个拟南芥和12个水稻WOX基因家族成员可分为3类,其中第3类为苦荞独有。基因组共线性分析表明,6个WOX基因在苦荞和拟南芥之间存在基因组共线性。表达模式及相关性表明,FtWOX1/FtWOX12/FtWOX22/FtWOX23/FtWOX24与苦荞出愈率存在正相关性。【结论】苦荞FtWOXs成员存在丰富的序列变异特征,不同苦荞基因型中WOX基因表达水平及出愈率存在明显差异和一定的相关性,揭示不同苦荞WOX基因具有潜在的功能多样性。

关键词: 苦荞, WOX基因, 愈伤组织, 基因表达

Abstract:

【Objective】 This study aimed to identify the whole genome WOX (WUSCHEL-related home obox) gene family in Tartary buckwheat and reveal the correlation with sequence characteristics of its gene family members, gene expression pattern and the rate of callus induction. It provides a theoretical basis for breaking through the regeneration and genetic transformation problem of Tartary buckwheat. 【Method】 The protein and nucleic acid sequence of the WOX gene family members in Tartary buckwheat were obtained by homology blast and the sequence of Arabidopsis WOX genes were served as reference. Based on protein homology and conserved domain analysis, all members of Tartary buckwheat WOX gene family were identified. The TBtools software was used to further demonstrate the characteristics of the WOX genes in Tartary buckwheat, including gene structure, conserved domain and cis-acting element. Genomic collinearity of WOX gene family members between Tartary buckwheat and Arabidopsis thaliana was analysed. Based on proximity method, the MEGA X software was used to perform phylogenetic tree of these WOX genes in Tartary buckwheat, Arabidopsis and rice. The hypocotyl explants of 70 Tartary buckwheat varieties were cultured with MS+2,4-D 3.0 mg·L ^-1+6-BA 1.0 mg·L^-1 for callus induction and the callus emergence rate of different genotypes was evaluated. The FtWOX gene expression level was performed by qPCR to compare the different Tartary buckwheat varieties with high and low callus yield. The correlation between callus rate and FTWOXS gene family members was analysed based on Pearson correlation coefficient. 【Result】 A total of 30 WOX genes were identified in Tartary buckwheat and they were unevenly distributed on 8 chromosomes. The 30 Tartary buckwheat WOX genes could be divided into three groups by phylogenetic tree. The WOX genes contained different conserved domains in different groups, and the main conserved domains were HD(Homeodomain), START and MEKHLA. The conserved motif analysis showed that the conserved motif number of FtWOX genes may contain 2 to 10 motifs, and the gene structure analysis showed that the number of exons contained in the genes between 2 to 18. Promoter elements analysis showed 26 different kinds of cis-acting elements in the 30 WOX genes. The phylogenetic analysis showed that 30 Tartary buckwheat, 15 Arabidopsis thaliana and 12 rice WOX gene family members could be divided into three categories, of which the third group is unique to Tartary buckwheat. The collinearity analysis showed that six WOX genes were genomic collinearity between Tartary buckwheat and Arabidopsis thaliana. Expression pattern and correlation analysis show that the expression level of FtWOX1/FtWOX12/FtWOX22/FtWOX23/ FtWOX24 has positive correlation with the callus induction. 【Conclusion】 Collectively, these data suggest that the Tartary buckwheat FtWOX members showed abundant sequence variation characteristics. The expression level and callus rate of WOX gene in different Tartary buckwheat genotypes were significantly different and correlated to some extent, suggesting that different Tartary buckwheat WOX genes had potential functional diversity.

Key words: Tartary buckwheat, WOX gene family, callus induction, gene expression

侯思宇,王欣芳,杜伟,冯晋华,韩渊怀,李红英,刘龙龙,孙朝霞. 苦荞WOX家族全基因组鉴定及响应愈伤诱导率表达分析[J]. 中国农业科学, 2021, 54(17): 3573-3586.

HOU SiYu,WANG XinFang,DU Wei,FENG JinHua,HAN YuanHuai,LI HongYing,LIU LongLong,SUN ZhaoXia. Genome-Wide Identification of WOX Family and Expression Analysis of Callus Induction Rate in Tartary Buckwheat[J]. Scientia Agricultura Sinica, 2021, 54(17): 3573-3586.

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来源Origin	品种Variety name
中国湖北Hubei, China	ZNQ171, ZNQ164, ZNQ165, ZNQ166, ZNQ167, ZNQ168, ZNQ169, ZNQ170
中国贵州Guizhou, China	ZNQ183, ZNQ184, ZNQ185, ZNQ186, ZNQ187, ZNQ188, ZNQ189
中国广西Guangxi, China	ZNQ194, ZNQ195, ZNQ196, ZNQ197, ZNQ198
中国安徽Anhui, China	ZNQ161, ZNQ162, ZNQ163
中国云南Yunnan, China	ZNQ192, ZNQ190, ZNQ191
中国甘肃Gansu, China	ZNQ154, ZNQ155, ZNQ156
中国青海Qinghai, China	ZNQ158, ZNQ159
中国湖南Hunan, China	ZNQ176, ZNQ177, ZNQ178
美国USA	PI647612, PI476852, PI658431, PI658429, PI658439, PI647613
不丹Bhutan	PI481673, PI481675, PI481658, PI481672
尼泊尔Nepal	PI427240, PI481666, PI658430
未知Unknown	ZNQ172, ZNQ174, ZNQ180, PI673868, PI673869, PI673870, PI673874, PI673858, PI673876, PI673845, PI673856, PI673857, PI673855, PI673862, PI673854, PI673846, PI673873, PI673863, PI673847, PI481673, PI673865, PI658438, PI673852

引物名称 Primer name	正向引物 Forward sequences (5'-3')	反向引物 Revered sequences (5'-3')	扩增长度 Amplification length (bp)
FtWOX1	CAGGCGGTGTCTTCTCGATT	AACTGACCGGAAGATGGGTG	94
FtWOX2	GATGAAGGACGACGAGCCAG	AGAACTCGGTAGGGTTGCAG	137
FtWOX3	CGAGGGGAACGATAAGGTGG	GACTCAACTTTGCCATGCTCC	142
FtWOX4	CTCAAGCGCCTTACTCCCTC	GCTTTCAAACGCCCTACTCG	102
FtWOX5	AGGACCTGCAAATGGGAAGC	TCTGAGACGGCTGCTACTCT	138
FtWOX6	AAGGTGCTCTAATGGCCGC	TTGGTCAACGCTTAAACGCC	137
FtWOX7	TTCAAGTCCAAGCCCTGCAA	CTCCGCTGTCGATCCATCTC	108
FtWOX8	ACACGACTCTCTCCTGCTTG	TGTCGTTGGACTGTTGTGGT	142
FtWOX9	AGTGGACAGTTCGTTGCTGA	GGACTCCTGACCGTCGTAAC	133
FtWOX10	ACTACATTGGCACCTGGTCG	CCACTAAGCTGCCATCCTCC	124
FtWOX11	AGGCGTGCTAGATTGAAGAGG	CTTGTGGCTGGGAATCCTCG	124
FtWOX12	AGAGGACCACCATAGACTCG	ACTGAACCGGACCCTCATTC	98
FtWOX13	ACCTTCCAATCAGTTCGCGG	ACTTCAAGTTGCTCGTCGGT	117
FtWOX14	GGTGGAACCCTACGCAAGAG	CTGAGTTGCGTCGTGATGTG	106
FtWOX15	GGTAGTCCGACTTGAAGGGC	CAACCGCCAACTGCATTCTC	111
FtWOX16	AGGCGTGCTAGATTGAAGAGG	CTTGTGGCTGGGAATCCTCG	124
FtWOX17	CCTCGAGAAGAGCTTCGACA	AACCAGATAGCAACCTGCCG	102
FtWOX18	ACCAAGCTTGAACCACGTCA	AGCTGCTTCGACTTCCATCG	113
FtWOX19	ACGGAGATGGAGTGCGAGTA	GTAGAGCTCGGCACAGTCAA	128
FtWOX20	TCTCACAGAGCAGAATCGCTG	GTCCTTGAATGGCAGGAGGC	129
FtWOX21	GCGGCATTGCGTCATCTAAG	TTCAACCTCTGGCTGAGTGC	107
FtWOX22	TCTTGCCCGTTGGATCACTC	GCAACACAAGATGGCATCCG	127
FtWOX23	CGTGATTGCCGAGCTGTAGA	AGCCGATGCCAAAGTCGTAA	108
FtWOX24	TGAGGAGAAGTCTCCAGGGT	CCGGTTCAGGCCTCATCATA	104
FtWOX25	CGTGAACAGGAGATGCCGAT	TACTGAACTTGCGTCGGTGT	120
FtWOX26	TGTGAAGAAAGCTCCCTCGG	TTCTGTATTTGCTGCGCCGT	111
FtWOX27	CATACGATGACTCTCCAGGGC	ATCTAGCGGCGGATGTTCG	116
FtWOX28	TCAGCGATGAGCAGATCAGG	CATCTGGCTCGTCGGTTCTG	145
FtWOX29	AAGCTTGCTCGTGAGCTTGG	ATGGAGACGGCATCGAACTC	134
FtWOX30	TACGCTCCGGTTGACATTCC	AGGCAACAGAGAGAAGCGAC	130
FtHis	ATCGACTGGAGGAAAGGCTC	GCGGTATCTGTGGGACTTCT	106