中国农业科学 ›› 2021, Vol. 54 ›› Issue (17): 3573-3586.doi: 10.3864/j.issn.0578-1752.2021.17.002
侯思宇1(),王欣芳1,杜伟1,冯晋华1,韩渊怀1,李红英1,刘龙龙2,孙朝霞1()
收稿日期:
2021-02-18
接受日期:
2021-05-08
出版日期:
2021-09-01
发布日期:
2021-09-09
通讯作者:
孙朝霞
作者简介:
侯思宇,Tel:18635068055;E-mail: 基金资助:
HOU SiYu1(),WANG XinFang1,DU Wei1,FENG JinHua1,HAN YuanHuai1,LI HongYing1,LIU LongLong2,SUN ZhaoXia1()
Received:
2021-02-18
Accepted:
2021-05-08
Online:
2021-09-01
Published:
2021-09-09
Contact:
ZhaoXia SUN
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。
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.
表1
用于愈伤组织诱导的苦荞品种"
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 |
表2
实时荧光定量PCR引物序列"
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 |
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