YIN Shu-xin,JI Jun-jie,XIAO Qing,et al.Cloning and Plant Expression Vector Construction of Drought-Induced GmNF-YA7[J].Soybean Science,2022,41(01):43-048.[doi:10.11861/j.issn.1000-9841.2022.01.0043]
干旱诱导基因GmNF-YA7克隆及植物表达载体构建
- Title:
- Cloning and Plant Expression Vector Construction of Drought-Induced GmNF-YA7
- Keywords:
- soybean; GmNF-YA7; drought response; plant expression vector; genetically engineered strain
- 文献标志码:
- A
- 摘要:
- 为了鉴定大豆核因子YA（Nuclear Factor YA， NF-YA）与非生物胁迫的关系，本研究检测大豆干旱胁迫下GmNF-YA7和GmNF-YA8的表达量情况，克隆GmNF-YA7基因并构建植物表达载体，同时获得其转基因工程菌株。qRT-PCR结果显示，GmNF-YA7和GmNF-YA8均可以被干旱胁迫诱导表达，且GmNF-YA7对干旱胁迫的应答更明显。从大豆中克隆出GmNF-YA7基因，其位于大豆8号染色体上，编码含有336个氨基酸的蛋白质，预测分子量为37.06 kDa，pI6.11。GmNF-YA7蛋白氨基酸序列中含有1个CBF保守结构域。亚细胞定位预测结果显示，GmNF-YA7定位于细胞核中。蛋白系统进化分析表明GmNF-YA7蛋白与拟南芥AtNF-YA1蛋白的亲缘关系较近。利用限制性内切酶Nde I和Sal I将GmNF-YA7与植物表达载体pRI101连接并转化农杆菌EHA105，获得转基因工程菌株。
- Abstract:
- In order to identify the relationship between soybean nuclear factor YA (NF-YA) and abiotic stress， we detected the expression levels of GmNF-YA7 and GmNF-YA8 in soybean under drought stress， cloned the GmNF-YA7， constructed the plant expression vector， and obtained genetically engineered strain in this reaserch. qRT-PCR results showed that both expression levels of GmNF-YA7 and GmNF-YA8 could be induced by drought stress， and the response of GmNF-YA7 to drought stress was more obvious. GmNF-YA7 was cloned from soybean， it was located on chromosome 8 of soybean and encoded a protein containing 336 amino acids. The predicted molecular weight of GmNF-YA7 protein was 37.06 kDa and the pI was 611. The amino acid sequence of GmNF-YA7 protein contained a conserved CBF domain. Subcellular localization prediction results showed that GmNF-YA7 protein was localized in the nucleus. Phylogenetic analysis showed that GmNF-YA7 protein was closely related to Arabidopsis AtNF-YA1 protein. GmNF-YA7 was constructed into plant expression vector pRI101 with restriction endonucleases Nde I and Sal I and transformed into Agrobacterium EHA105. This study showed that the GmNF-YA7 gene was up regulated by drought， and we constructed plant expression vector with GmNF-YA7 and genetically obtained engineered Agrobacterium strain with this vector.
参考文献/References:
［1］LEE H， FISCHER R L， GOLDBERG R B， et al. Arabidopsis LEAFY COTYLEDON1 represents a functionally specialized subunit of the CCAAT binding transcription factor[J]. Proceedings of the National Academy of Sciences of the United States of America， 2003， 100(4): 2152-2156.[2]MANTOVANI R. The molecular biology of the CCAAT-binding factor NF-Y[J]. Gene， 1999， 239(1): 15-27.[3]CERIBELLI M， DOLFINI D， MERICO D， et al. The histone-like NF-Y is a bifunctional transcription factor[J]. Molecular and Cellular Biology， 2008， 28(6): 2047-2058.[4]KWONG R W， BUI A Q， LEE H， et al. LEAFY COTYLEDON1-LIKE defines a class of regulators essential for embryo development[J]. The Plant Cell， 2003， 15(1): 5-18.[5]WARPEHA K M， UPADHYAY S， YEH J， et al. The GCR1， GPA1， PRN1， NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis[J]. Plant Physiology， 2007， 143(4): 1590-1600.[6]李娟， 高凯， 安新民. 转录因子NF-Y在植物生长发育和逆境胁迫响应中的作用[J]. 中国细胞生物学学报， 2019， 41(12): 188-196. (LI J， GAO K， AN X M， et al. Roles of transcription factor NF-Y in plant growth， development and response to stress[J]. Chinese Journal of Cell Biology， 2019， 41(12): 188-196.)[7]PETRONI K， KUMIMOTO R W， GNESUTTA N， et al. The pro-miscuous life of plant NUCLEAR FACTOR Y transcription factors[J]. The Plant Cell， 2012， 24(12): 4777-4792.[8]LI W X， OONO Y， ZHU J， et al. The Arabidopsis NFYA5 trans-cription factor is regulated transcriptionally and post transc-riptionally to promote drought resistance[J]. The Plant Cell， 2008， 20(8): 2238-2251.[9]LEE D K， KIM H， JANG G， et al. The NF-YA transcription factor OsNF-YA7 confers drought stress tolerance of rice in an abscisic acid independent manner[J]. Plant Science， 2015， 241: 199-210.[10]LUAN M， XU M， LU Y， et al. Family-wide survey of miR169s and NF-YAs and their expression profiles response to abiotic stress in maize roots[J]. PLoS One， 2014， 9(3): e91369.[11]SU H， CAO Y， KU L， et al. Dual functions of ZmNF-YA3 in photoperiod-dependent flowering and abiotic stress responses in maize[J]. Journal of Experimental Botany， 2018， 69(21): 5177-5189. [12]LIAN C， LI Q， YAO K， et al. Populus trichocarpa PtNF-YA9， a multifunctional transcription factor， regulates seed germination， abiotic stress， plant growth and development in Arabidopsis[J]. Frontiers in Plant Science， 2018， 9: 954. [13]杨如萍， 包振贤， 陈光荣， 等. 大豆抗旱性研究进展[J]. 作物杂志， 2012（5）: 8-12. (YANG R P， BAO Z X， CHEN G R， et al. The research progress in drought resistance of soybean[J]. Crops， 2012（5）: 8-12.)[14]QUACH T N， NGUYEN H T， VALLIYODAN B， et al. Genome-wide expression analysis of soybean NF-Y genes reveals potential function in development and drought response[J]. Molecular Genetics and Genomics， 2015， 290(3): 1095-1115.[15]NI Z， HU Z， JIANG Q， et al. GmNFYA3， a target gene of miR169， is a positive regulator of plant tolerance to drought stress[J]. Plant Molecular Biology， 2013， 82(1-2): 113-129.[16]QIU S， ZHANG J， HE J， et al. Overexpression of GmGolS2-1， a soybean galactinol synthase gene， enhances transgenic tobacco drought tolerance[J]. Plant Cell Tissue and Organ Culture， 2020， 143(3): 507-516. [17]何佳琦， 翟莹， 张军， 等. 大豆转录因子GmDof1.5的克隆及非生物胁迫诱导表达[J]. 浙江农业学报， 2021， 33(1): 1-7. (HE J Q， ZHAI Y， ZHANG J， et al. Cloning and expression analysis of GmDof1.5 in soybean under abiotic stress[J]. Acta Agriculturae Zhejiangensis， 2021， 33(1): 1-7.)[18]LIVAK K J， SCHMITTGEN T D. Analysis of relative gene exp-ression data using real-time quantitative PCR and the 2 -ΔΔCT method[J]. Methods， 2001， 25(4): 402-408. [19]丁慧霞， 刘凤， 张利娟， 等. 植物中NF-Y转录因子的结构和功能研究进展[J]. 分子植物育种， 2017， 15(5): 105-115. (DING H X， LIU F， ZHANG L J， et al. The structure and function of NF-Y in plants[J]. Molecular Plant Breeding， 2017， 15(5): 1691-1701.)[20]方广宁， 胡利芹， 王二辉， 等. 谷子转录因子SiNF-YA6的过表达提高转基因植株对低氮胁迫的抗性[J]. 中国农业科学， 2015， 48(20): 3989-3997. (FANG G N， HU L Q， WANG E H， et al. Overexpression of a transcription factor gene SiNF-YA6 from millet (Setaria italica) enhanced the resistance of transgenic plants to nitrogen starvation[J]. Scientia Agricultura Sinica， 2015， 48(20): 3989-3997.)[21]LI Y J， FANG Y， FU Y R， et al. NFYA1 is involved in reg-ulation of postgermination growth arrest under salt stress in Arabidopsis[J]. PLoS One， 2013， 8(4): e61289.[22]黄锁， 胡利芹， 徐东北， 等. 谷子转录因子SiNF-YA5通过ABA非依赖途径提高转基因拟南芥耐盐性[J]. 作物学报， 2016， 42(12): 1787-1797. (HUANG S， HU L Q， XU D B， et al. Transcription factor SiNF-YA5 from foxtail millet (Setaria italica) conferred tolerance to high-salt stress through ABA-independent pathway in transgenic Arabidopsis[J]. Acta Agronomica Sinica， 2016， 42(12): 1787-1797.)
相似文献/References:
[1]刘章雄,李卫东,孙石,等.1983～2010年北京大豆育成品种的亲本地理来源及其遗传贡献[J].大豆科学,2013,32(01):1.[doi:10.3969/j.issn.1000-9841.2013.01.002]
LIU Zhang-xiong,LI Wei-dong,SUN Shi,et al.Geographical Sources of Germplasm and Their Nuclear Contribution to Soybean Cultivars Released during 1983 to 2010 in Beijing[J].Soybean Science,2013,32(01):1.[doi:10.3969/j.issn.1000-9841.2013.01.002]
[2]李彩云,余永亮,杨红旗,等.大豆脂质转运蛋白基因GmLTP3的特征分析[J].大豆科学,2013,32(01):8.[doi:10.3969/j.issn.1000-9841.2013.01.003]
LI Cai-yun,YU Yong-liang,YANG Hong-qi,et al.Characteristics of a Lipid-transfer Protein Gene GmLTP3 in Glycine max[J].Soybean Science,2013,32(01):8.[doi:10.3969/j.issn.1000-9841.2013.01.003]
[3]王明霞,崔晓霞,薛晨晨,等.大豆耐盐基因GmHAL3a的克隆及RNAi载体的构建[J].大豆科学,2013,32(01):12.[doi:10.3969/j.issn.1000-9841.2013.01.004]
WANG Ming-xia,CUI Xiao-xia,XUE Chen-chen,et al.Cloning of Halotolerance 3 Gene and Construction of Its RNAi Vector in Soybean (Glycine max)[J].Soybean Science,2013,32(01):12.[doi:10.3969/j.issn.1000-9841.2013.01.004]
[4]张春宝,李玉秋,彭宝,等.线粒体ISSR与SCAR标记鉴定大豆细胞质雄性不育系与保持系[J].大豆科学,2013,32(01):19.[doi:10.3969/j.issn.1000-9841.2013.01.005]
ZHANG Chun-bao,LI Yu-qiu,PENG Bao,et al.Identification of Soybean Cytoplasmic Male Sterile Line and Maintainer Line with Mitochondrial ISSR and SCAR Markers[J].Soybean Science,2013,32(01):19.[doi:10.3969/j.issn.1000-9841.2013.01.005]
[5]卢清瑶,赵琳,李冬梅,等.RAV基因对拟南芥和大豆不定芽再生的影响[J].大豆科学,2013,32(01):23.[doi:10.3969/j.issn.1000-9841.2013.01.006]
LU Qing-yao,ZHAO Lin,LI Dong-mei,et al.Effects of RAV gene on Shoot Regeneration of Arabidopsis and Soybean[J].Soybean Science,2013,32(01):23.[doi:10.3969/j.issn.1000-9841.2013.01.006]
[6]杜景红,刘丽君.大豆fad3c基因沉默载体的构建[J].大豆科学,2013,32(01):28.[doi:10.3969/j.issn.1000-9841.2013.01.007]
DU Jing-hong,LIU Li-jun.Construction of fad3c Gene Silencing Vector in Soybean[J].Soybean Science,2013,32(01):28.[doi:10.3969/j.issn.1000-9841.2013.01.007]
[7]张力伟,樊颖伦,牛腾飞,等.大豆“冀黄13”突变体筛选及突变体库的建立[J].大豆科学,2013,32(01):33.[doi:10.3969/j.issn.1000-9841.2013.01.008]
ZHANG Li-wei,FAN Ying-lun,NIU Teng-fei?,et al.Screening of Mutants and Construction of Mutant Population for Soybean Cultivar "Jihuang13”[J].Soybean Science,2013,32(01):33.[doi:10.3969/j.issn.1000-9841.2013.01.008]
[8]盖江南,张彬彬,吴瑶,等.大豆不定胚悬浮培养基因型筛选及基因枪遗传转化的研究[J].大豆科学,2013,32(01):38.[doi:10.3969/j.issn.1000-9841.2013.01.009]
GAI Jiang-nan,ZHANG Bin-bin,WU Yao,et al.Screening of Soybean Genotypes Suitable for Suspension Culture with Adventitious Embryos and Genetic Transformation by Particle Bombardment[J].Soybean Science,2013,32(01):38.[doi:10.3969/j.issn.1000-9841.2013.01.009]
[9]王鹏飞,刘丽君,唐晓飞,等.适于体细胞胚发生的大豆基因型筛选[J].大豆科学,2013,32(01):43.[doi:10.3969/j.issn.1000-9841.2013.01.010]
WANG Peng-fei,LIU Li-jun,TANG Xiao-fei,et al.Screening of Soybean Genotypes Suitable for Somatic Embryogenesis[J].Soybean Science,2013,32(01):43.[doi:10.3969/j.issn.1000-9841.2013.01.010]
[10]刘德兴,年海,杨存义,等.耐酸铝大豆品种资源的筛选与鉴定[J].大豆科学,2013,32(01):46.[doi:10.3969/j.issn.1000-9841.2013.01.011]
LIU De-xing,NIAN Hai,YANG Cun-yi,et al.Screening and Identifying Soybean Germplasm Tolerant to Acid Aluminum[J].Soybean Science,2013,32(01):46.[doi:10.3969/j.issn.1000-9841.2013.01.011]
备注/Memo
收稿日期：2021-08-26