葡萄生长调控因子GRF家族基因的鉴定及表达分析

doi:10.16420/j.issn.0513-353x.2021-0533

摘要/Abstract

摘要：

开展葡萄生长调控因子GRF（Growth-regulating factor）家族成员的基因结构、蛋白保守基序、亚细胞定位预测、同线性、系统进化树及顺式作用元件等分析;利用qRT-PCR技术开展有核和无核葡萄不同组织器官、种子发育不同时期及激素响应表达模式分析。葡萄GRF家族共有8个成员,其中7个分布于6条染色体,编码氨基酸数为213 ~ 604,所有成员均定位于细胞核。根据进化关系分为4组（A ~ D）,同组VvGRF的外显子—内含子结构、保守基序数和类型均具有保守性。所有葡萄GRF蛋白N端均含有QLQ和WRC保守结构域,C端至少含有TQL或FFD结构域之一。同线性分析发现,VvGRF3和VvGRF4存在并联重复。VvGRF启动子区发现大量与生长发育、激素响应及胁迫响应相关顺式作用元件。大部分VvGRF在营养器官叶片中高表达,VvGRF2在生殖器官花和果实中高表达;VvGRF3和VvGRF6在无核葡萄种子发育过程中表达量显著高于有核葡萄,而VvGRF8在有核葡萄种子发育前期表达量显著高于无核葡萄;VvGRF响应GA₃和IAA诱导,且大部分基因在处理0.5或1 h后下调。

关键词: 葡萄, GRF, 家族进化, 种子发育, GA₃, IAA, 表达分析

Abstract:

The gene structure analysis,protein conserved motif identification,subcellular localization prediction,synteny analysis,phylogenetic analysis,and cis-acting elements analysis of Growth-regulating factor（GRF）family members were performed. Expression patterns of grape GRF genes in different plant parts and against different hormone applications were analyzed. Further expression patterns were compared during different stages of seed development in seeded and seedless cultivars. Eight grape GRF genes were identified and divided into four groups（A-D）. Seven of the genes were unevenly distributed on six chromosomes having and the number of amino acids was 213-604. All grape GRF proteins were predicted in the nucleus. Moreover,VvGRFs in the same clade showed conserved exon-intron structure and motif distribution patterns. All GRF proteins contained QLQ and WRC domains in the N-terminal region and at least one TQL or FFD domain in the C-terminal region. Synteny analysis showed that VvGRF3 and VvGRF4 were segmentally duplicated. Various cis-acting elements related to growth and development,hormones response,and stresses were found in the promoter region of VvGRF genes. Expression analysis illustrated that most of the VvGRFs were highly expressed in vegetative organs i.e. leaves. However,VvGRF2 was highly expressed in reproductive organs like flowers and fruits. The expression of VvGRF3 and VvGRF6 was significantly higher during seed development in seedless grapes as compared to seeded grapes,while VvGRF8 showed high expression in seeded grapes. The expression of VvGRF genes was induced against GA₃ and IAA treatments,and most of the VvGRFs were down-regulated at 0.5 or 1 h.

Key words: grape, GRF, family evolution, seed development, GA₃, IAA, expression analysis

中图分类号:

S663.1

梁晨, 孙如意, 向锐, 孙艺萌, 师校欣, 杜国强, 王莉. 葡萄生长调控因子GRF家族基因的鉴定及表达分析[J]. 园艺学报, 2022, 49(5): 995-1007.

LIANG Chen, SUN Ruyi, XIANG Rui, SUN Yimeng, SHI Xiaoxin, DU Guoqiang, WANG Li. Genome-wide Identification of Grape GRF Family and Expression Analysis[J]. Acta Horticulturae Sinica, 2022, 49(5): 995-1007.

图/表 11

表1 用于qRT-PCR反应的引物序列

Table 1 The primer sequences used for qRT-PCR amplification

基因Gene	上游引物（5'-3'）Forward primer	下游引物（5'-3'）Reverse primer
VvGRF1	GGGCTTCCAGTGCCTTATC	TTCCCATCCGTTCTTCTACAT
VvGRF2	CTGGTGGGACTCATTTCGC	AGGTTGCCTGGCTGTTATTG
VvGRF3	GCCTCAATGACTTCTGAAACCT	CGATGATGCCTGTAATCCCTAT
VvGRF4	CAGAAAGCCTGTGGAAGTTATG	TGTAGCAAGAAGGGTGACGAT
VvGRF5	CGCCTTTGGACAGCACAT	CGAGACTGACCCTCCTGTTTC
VvGRF6	TCGTTCTATCTTCAACCTCCGT	AGTACTTCTGATTGAGTGCCACAT
VvGRF7	GAAGCCCTTGATTCCGTTG	TCTCACAATACTTCTGATCGGC
VvGRF8	CGCCACTCAGGCTATCTCG	AAGCAGGCTCCCATGTTGT
EF1-α	AGGAGGCAGCCAACTTCACC	CAAACCCTGCATCACCATTC

表2 葡萄基因组中的GRF家族基因信息

Table 2 Information of GRF family gene in grape genome

基因 Gene	登录号 Accession No.	基因ID Gene locus ID	VCost.v3 ID	起始与终止位点/bp Start and end position	CDS/bp	ORF/ aa	亚细胞定位 Subcellular localization
VvGRF1	XP_002281639.1	GSVIVT01019913001	Vitvi02g00449	4 421 518-4 424 601	639	213	细胞核Nucleus
VvGRF2	XP_002277233.3	GSVIVT01033800001	Vitvi08g01498	17 725 858-17 728 519	1 176	392	细胞核Nucleus
VvGRF3	XP_010654438.1	GSVIVT01016762001	Vitvi09g00107	1 135 208-1 137 036	1 092	364	细胞核Nucleus
VvGRF4	XP_010656126.2	GSVIVT01015095001	Vitvi11g00092	1 000 359-1 001 747	1 164	388	细胞核Nucleus
VvGRF5	XP_002270427.2	GSVIVT01024326001	Vitvi16g00073	975 755-979 958	1 122	374	细胞核Nucleus
VvGRF6	XP_002271355.3	GSVIVT01038629001	Vitvi16g01354	21 346 554-21 349 482	1 812	604	细胞核Nucleus
VvGRF7	CBI19365.3	GSVIVT01009299001	Vitvi18g00623	7 086 633-7 089 964	1 428	476	细胞核Nucleus
VvGRF8	CBI33069.3	GSVIVT01007165001	Vitvi07g01551	30 596 800-30 600 327	1 674	558	细胞核Nucleus

图1 葡萄GRF基因同线性分析彩带连接的两个染色体区域为同线区域。

Fig. 1 Synteny analysis of grapevine GRF genes Colored lines connection two chromosomal regions are syntenic regions.

图2 葡萄GRF家族进化树（a）、基因结构（b）及蛋白保守基序（c）

Fig. 2 Phylogenetic tree（a）,gene structure（b）and conserved domain（c）of grapevine GRF family

图3 葡萄与其他物种GRF家族的系统进化树

Fig. 3 Phylogenetic analysis of GRF family in grape and other plants

图4 葡萄GRF家族基因启动子顺式作用元件分析

Fig. 4 Analysis of cis-acting elements in the promoter of grape GRF family genes

图5 ‘无核白’和‘红地球’葡萄中GRF家族基因在不同器官中的表达（qRT-PCR）不同字母表示相同品种不同器官间差异显著,Tukey检验方差分析,P < 0.05。

Fig. 5 Expression of GRF family genes in‘Thompson Seedless’and‘Red Globe’grape in different organs（qRT-PCR） Different letters indicate statistically significant differences among different organs in same cultivar. ANOVA with a Tukey post-hoc analysis,P < 0.05.

图6 GRF基因在‘红地球’和‘森田尼无核’杂交分离有核（S）和无核（SL）子代种子发育时期的表达

Fig. 6 Expression of GRF genes in the‘Red Globe’and‘Centennial Seedless’hybridization separation seeded（S）and seedless（SL） progenies during seed development

图7 GRF基因在有核和无核葡萄种子发育过程中的表达不同字母表示相同天数不同品种间差异显著,Tukey检验方差分析,P < 0.05。

Fig. 7 Expression of GRF genes in seeded and seedless grapes during seed development Different letters indicate significant differences among different cultivars in the same day. ANOVA with a Tukey post-hoc analysis,P < 0.05.

图8 qRT-PCR分析GA3处理后葡萄GRF家族基因在叶片中的表达

Fig. 8 qRT-PCR analysis of grape GRF family genes in grape leaves treated with GA3 t-test（* α = 0.05,** α = 0.01）.

图9 qRT-PCR分析IAA处理后葡萄GRF家族基因在叶片中的表达

Fig. 9 qRT-PCR analysis of grape GRF family genes in grape leaves treated with IAA t-test（* α = 0.05,** α = 0.01）.

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