[1]宋新新,袁月,孙海龙,等.葡萄VvGW2基因的克隆及表达[J].江苏农业学报,2015,(02):434-440.[doi:10.3969/j.issn.1000-4440.2015.02.033]
　SONG Xin-xin,YUAN Yue,SUN Hai-long,et al.Cloning and expression of VvGW2 gene in grape[J].,2015,(02):434-440.[doi:10.3969/j.issn.1000-4440.2015.02.033]
点击复制

葡萄VvGW2基因的克隆及表达()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2015年02期
页码:
434-440
栏目:
园艺
出版日期:
2015-04-30

文章信息/Info

Title:
Cloning and expression of VvGW2 gene in grape
作者:
宋新新; 袁月; 孙海龙; 余晓娟; 陶建敏
(南京农业大学园艺学院，江苏南京210095)
Author(s):
SONG Xin-xin; YUAN Yue; SUN Hai-long; YU Xiao-juan; TAO Jian-min
(College of Horticulture, Nanjing Agricultural University, Nanjing 210095，China)
关键词:
葡萄; VvGW2基因; 基因克隆; 表达分析
Keywords:
grape; VvGW2 gene; gene cloning; expression analysis
分类号:
S663.1
DOI:
10.3969/j.issn.1000-4440.2015.02.033
文献标志码:
A
摘要:
为了从葡萄品种美人指中克隆VvGW2基因，并对其结构特征及表达模式进行分析。在NCBI中查找与水稻粒形基因OsGW2同源性最高的葡萄序列，根据葡萄的GW2基因序列设计特异引物。采用改良CTAB法提取美人指花序中总RNA，运用RT-PCR技术进行克隆。利用NCBI数据库中的BLASTn和BLASTp程序进行相似性分析；利用Bioxm2.6推测分析蛋白质分子量和等电点；根据NCBI中Conserved Domains程序进行蛋白质保守域结构预测；用ExPaSy提供的在线SOPMA程序进行蛋白质二级结构预测；利用实时荧光定量RT-PCR研究该基因表达模式。结果显示，从美人指中克隆得到1个GW2基因同源序列，命名为VvGW2基因。VvGW2基因开放阅读框长度为1 272 bp，共编码423个氨基酸，预测蛋白质分子量为46 960，理论等电点为4.68。该基因编码的氨基酸具有GW2蛋白质保守的环指结构域，该环指蛋白质为C5HC2类型。与GenBank中登录的其他植物GW2蛋白质序列相似性为47%～53%。根据VvGW2基因所编码的氨基酸序列构建系统进化树，结果显示，葡萄与可可聚为一类。实时荧光定量RT-PCR分析结果显示，VvGW2基因在美人指花或果实的各时期均有表达，其中在开花期基因表达量最高。不同葡萄品种中，VvGW2基因的表达量存在差异，在指形葡萄品种美人指的表达量最高，在圆形葡萄品种中的表达量很低。表明VvGW2基因在不同时期和不同品种中的表达量差异可能与葡萄果实形状相关。
Abstract:
To isolate the VvGW2 gene from grape (Vitis vinifera) cultivar Manicure Finger, and analyze the structure and expression pattern of the gene, specific primers were designed based on the grape sequence in NCBI, the highest homologue with the rice grain shape gene OsGW2. The improved CTAB method was used to isolate total RNA and the VvGW2 gene was obtained using RT-PCR. BLASTn and BLASTp in NCBI were used to perform the similarity analysis, the protein molecular weight and isoelectric point were speculated using Bioxm2.6, the conserved domain structure of protein was predicted by Conserved Domains program in NCBI, the protein secondary structure was predicted by using SOPMA program. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern of GW2. The VvGW2 gene contains a 1 272-bp open reading frame (ORF) which encodes 423 amino acids with a calculated molecular weight of 46 960 and an isoelectric point of 4.68. Protein structure analysis showed that VvGW2 contained a RING domain and belonged to C5HC2. Similarity analysis showed that the prediced amino acid sequence of VvGW2 shared homologies of 53%, 48%, 48%, 48% and 47% with wheat, maize, sorghum, barly and rice, respectively. The phylogenetic tree constructed based on amino acid sequence of VvGW2 gene revealed that grape was clustered together with cocoa. Quantitative real-time PCR analysis showed that VvGW2 gene was expressed at each developmental stage of Manicure Finger, and the highest expression took place at anthesis. The expression level of VvGW2 gene varied in different cultivars, the highest in Manicure Finger, and the lowest in round shape grape. The fruit shape of grape might be responsible for the differential expression of VvGW2 at different developmental stages and in different cultivars.

参考文献/References:


[1]程云，吴欣欣，李百健，等．外源脱落酸对魏可葡萄果实着色及品质的影响[J]．江苏农业科学，2014，42(10)：163-166.
[2]芮东明，刘亚柏，王敬根，等．夏黑葡萄标准化栽培技术[J]．江苏农业科学，2014，42(8)：162-164.
[3]张海宁，王亚超，马辉，等．GC-MS分析夏黑葡萄中挥发性香气成分[J]．江苏农业科学，2014，42(8)：294-297.
[4]SONG X J, HUANG W, SHI M, et al. A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase[J]. Nature Genetics, 2007 (39): 623-630.
[5]HATAKEYAMA S, YADA M, MATSUMOTO M, et al. U box proteins as a new family of ubiquitin-protein ligases[J]. Journal of Biological Chemistry, 2001, 276(35): 33111-33120.
[6]BORDEN K L B, FREEMONT P S. The RING finger domain: a recent example of a sequence-structure family[J]. Current Opinion in Structural Biology, 1996, 6(3): 395-401.
[7]SAURIN A J, BORDEN K L B, BODDY M N, et al. Does this have a familiar RING?[J]. Trends in Biochemical Sciences, 1996, 21(6): 208-214.
[8]STONE S L, HAUKSDTTIR H, TROY A, et al. Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis[J]. Plant Physiology, 2005, 137(1): 13-30.
[9]杨东叶, 刘凯于, 余泽华. 泛素连接酶E3[J]. 中国细胞生物学学报, 2005, 27(3):281-285.
[10]XIE Q, GUO H S, DALLMAN G, et al. SINAT5 promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals[J]. Nature, 2002, 419(6903): 167-170.
[11]SAIJO Y, SULLIVAN J A, WANG H, et al. The COP1-SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity[J]. Genes & Development, 2003, 17(21): 2642-2647.
[12]DORNAN D, WERTZ I, SHIMIZU H, et al. The ubiquitin ligase COP1 is a critical negative regulator of p53[J]. Nature, 2004, 429(6987): 86-92.
[13]DISCH S, ANASTASIOU E, SHARMA V K, et al. The E3 ubiquitin ligase BIG BROTHER controls arabidopsis organ size in a dosage-dependent manner[J]. Current Biology, 2006, 16(3): 272-279.
[14]郑焕,张计育,王新卫,等. 葡萄雌能花相关基因VvMS2的克隆及表达分析[J]. 中国农业科学, 2013, 46(14): 2953-2962.
[15]LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods, 2001,25(4): 402-408.
[16]汤程贻,殷晶晶,邹爱兰,等. 紫草LeEXT-1基因的克隆、序列及表达分析[J]. 南京农业大学学报, 2013, 36(1): 13-18.
[17]LI Q, LI L, YANG X, et al. Relationship, evolutionary fate and function of two maize co-orthologs of rice GW2 associated with kernel size and weight[J]. BMC Plant Biology, 2010, 10(1): 143.
[18]刘方方,姜颖,曹言勇,等. 玉米中 RING 型 E3 泛素连接酶基因ZmGW2的表达分析[J]. 玉米科学, 2013, 21(2): 47-51.
[19]杨子博. 小麦粒重基因TaGW2的克隆, 原核表达及分子标记的开发[D]. 杨凌：西北农林科技大学, 2012.

相似文献/References:

[1]余阳,袁月,王继源,等.套袋及喷钙对魏可葡萄矿质元素和果实品质的影响[J].江苏农业学报,2015,(05):1134.[doi:doi:10.3969/j.issn.1000-4440.2015.05.030]
　YU Yang,YUAN Yue,WANG JI-yuan,et al.Effects of bagging treatments and calcium applications on mineral elements and fruit quality of Wink grape[J].,2015,(02):1134.[doi:doi:10.3969/j.issn.1000-4440.2015.05.030]
[2]王西成,吴伟民,巫建华,等.葡萄β-1,3-葡聚糖酶基因的克隆、序列分析及表达[J].江苏农业学报,2015,(04):899.[doi:10.3969/j.issn.1000-4440.2015.04.030]
　JP+WANG Xi-cheng,WU Wei-min,WU Jian-hua,et al.Cloning, characterization and expression analysis of beta-1,3-glucanase gene in grapevine[J].,2015,(02):899.[doi:10.3969/j.issn.1000-4440.2015.04.030]
[3]张杰,单宝来,田永生,等.葡萄HSP17基因的合成与功能分析[J].江苏农业学报,2017,(03):503.[doi:doi:10.3969/j.issn.1000-4440.2017.03.004]
　ZHANG Jie,SHAN Bao-lai,TIAN Yong-sheng,et al.Synthesis and functional analysis of HSP17 gene in grape[J].,2017,(02):503.[doi:doi:10.3969/j.issn.1000-4440.2017.03.004]
[4]冯娇,王武,侯旭东,等.基于转录组测序技术探究GA3和CPPU抑制葡萄果锈产生的机理[J].江苏农业学报,2017,(04):895.[doi:doi:10.3969/j.issn.1000-4440.2017.04.026]
　FENG Jiao,WANG Wu,HOU Xu-dong,et al.Underlying mechanism of GA3 and CPPU inhibiting grape fruit russet discovered based on transcriptome sequencing[J].,2017,(02):895.[doi:doi:10.3969/j.issn.1000-4440.2017.04.026]
[5]张波,姬长英,徐伟悦,等.不同预处理下无核厚皮葡萄热泵式分段干燥特性及品质比较[J].江苏农业学报,2018,(05):1144.[doi:doi:10.3969/j.issn.1000-4440.2018.05.025]
　ZHANG Bo,JI Chang-ying,XU Wei-yue,et al.Heat-pump stage-changed drying characteristics and product quality in comparison for seedless thick-skinned grapes under different pretreatment conditions[J].,2018,(02):1144.[doi:doi:10.3969/j.issn.1000-4440.2018.05.025]
[6]高世敏,董阳,王武,等.葡萄赤霉素合成关键基因VvGA20ox2的克隆、亚细胞定位和表达分析[J].江苏农业学报,2018,(06):1331.[doi:doi:10.3969/j.issn.1000-4440.2018.06.019]
　GAO Shi-min,DONG Yang,WANG Wu,et al.Cloning, subcellular localization and expression analysis of the key gene VvGA20ox2 in gibberellin synthesis of grapevine[J].,2018,(02):1331.[doi:doi:10.3969/j.issn.1000-4440.2018.06.019]
[7]雷旺雄,卢军.葡萄采摘机器人采摘点的视觉定位[J].江苏农业学报,2020,(04):1015.[doi:doi:10.3969/j.issn.1000-4440.2020.04.029]
　LEI Wang-xiong,LU Jun.Visual positioning method for picking point of grape picking robot[J].,2020,(02):1015.[doi:doi:10.3969/j.issn.1000-4440.2020.04.029]
[8]刘帅,张亚红,刘鑫,等.不同光源补光对设施红地球葡萄果实品质的影响[J].江苏农业学报,2021,(04):949.[doi:doi:10.3969/j.issn.1000-4440.2021.04.018]
　LIU Shuai,ZHANG Ya-hong,LIU Xin,et al.Effects of different supplementary light sources on fruit quality of Red Globe grape under protected cultivation[J].,2021,(02):949.[doi:doi:10.3969/j.issn.1000-4440.2021.04.018]
[9]高磊,李慧,洪奔,等.葡萄品种钟山红玉与钟山红杂交后代的SSR鉴定及葡萄果实性状遗传倾向分析[J].江苏农业学报,2023,(01):187.[doi:doi:10.3969/j.issn.1000-4440.2023.01.022]
　GAO Lei,LI Hui,HONG Ben,et al.SSR identification and genetic tendency analysis of fruit characters of hybrid offspring of Zhongshan Hongyu and Zhongshan Hong[J].,2023,(02):187.[doi:doi:10.3969/j.issn.1000-4440.2023.01.022]
[10]胡宏远,王静,李红英,等.调亏灌溉对贺兰山东麓赤霞珠葡萄主干茎流规律及品质的影响[J].江苏农业学报,2023,(03):798.[doi:doi:10.3969/j.issn.1000-4440.2023.03.021]
　HU Hong-yuan,WANG Jing,LI Hong-ying,et al.Effects of regulated deficit irrigation on stem flow and quality of Cabernet Sauvignon grape in eastern foot of Helan Mountain[J].,2023,(02):798.[doi:doi:10.3969/j.issn.1000-4440.2023.03.021]

备注/Memo

备注/Memo:
收稿日期：2014-09-17 基金项目：国家“948”重点项目（2011-G28） 作者简介：宋新新（1988- ），女，山东泰安人，硕士研究生，研究方向为果树生物技术。（Tel）15895996856；（E-mail）songxinxin2012@sina.com 通讯作者：陶建敏，（E-mail）tjm266@sina.com
更新日期/Last Update: 2015-04-30