Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (14): 2651-2661.doi: 10.3864/j.issn.0578-1752.2016.14.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

The Function Expression of Salt-Tolerant Yeast Gene Halotolerance ( HAL1 ) in Cotton

MU Min, SHU Na, WANG Shuai, GUO Li-xue, FAN Wei-li, YIN Zu-jun, WANG Jun-juan, WANG De-long, YE Wu-wei   

  1. Institute of Cotton Research, Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology/Key Laboratory for Cotton Genetic Improvement, Ministry of Agriculture, Anyang 455000, Henan
  • Received:2016-02-15 Online:2016-07-16 Published:2016-07-16

Abstract: 【Objective】 The objective of this study is to clone Saccharomyces cerevisiae halotolerance (ScHAL1) gene and transformed into cotton , explore the function of the gene in cotton, to further explore function of the salt-tolerant yeast genes in higher plants. 【Method】According to total length of mRNA sequence information of ScHAL1 in NCBI, the gene was cloned using RT-PCR technology from Saccharomyces cerevisiae As2.375, double enzyme digestion was made with XbaⅠand SmaⅠfor pBI121::GFP, and pBI121-ScHAL1::GFP fusion expression vector was constructed by In-Fusion technique. With weak auto-fluorescence upland cotton varieties, Y-2067, ZA-23 and GZ-2 pollen as materials, using the gene bombarding technique to study transient expression of cotton pollen. The expression vector pBI121-ScHAL1::GFP was transformed into cotton salt-sensitive material Zhong s9612 with gene gun in vivo conversion technology, T0 generation cotton genetically seeds were modified. Solution with 100 mmol·L-1 NaCl was used to test the salt resistance of seeds in the transgenic T0 generation seed germination experiment, molecular detection was carried out, and semal salt resistance of transgenic plants was analysis. 【Result】 ScHAL1 cloned from Saccharomyces cerevisiae As2.375 and ScHAL1 is 885 bp in length, which encoding 294 amino acids. After its sequence analysis, it was found that the largest proportion of the whole HAL1 protein is serine, and it is alkaline and positively charged and it is a hydrophilic protein. According to the results of protein secondary structure prediction, it is speculated that the structure of the protein function domain may mainly made up of random curl and beta sheet. Cotton pollen instantaneous expression results showed that after conversion of HAL1 gene, three land cotton powder green fluorescence were obviously enhanced, suggesting that the gene expressed in these three upland cotton pollen. With 100 mmol·L-1 NaCl, transgenic T0 generation seed germination ability obviously stronger than receptor s9612 selfing seed material, which preliminary showed that HAL1 could also improve the seed salt resistance. According to the gene nucleotide sequences, two pairs of primers were designed for molecular detection of T0 generation. Direct sequencing of PCR product was conducted and the sequencing results preliminarily evidence that the transgene is successful. With the semal salt resistance analysis, it was found that the chlorophyll contents of transgenic plants in 600 mmol·L-1 NaCl and 400 mmol·L-1 NaCl were higher than the control, and the chlorophyll contents of transgenic plants in 600 mmol·L-1 NaCl were higher than that in 400 mmol·L-1 NaCl. 【Conclusion】HAL1 gene was Successfully cloned from Saccharomyces cerevisiae, yeast HAL1 gene plays an important role in improving cotton salt resistance.

Key words: yeast, HAL1, upland cotton, pollen instantaneous expression, molecular detection

[1]    Boyer J S. Plant productivity and environment. American Association for the Advancement of Science, 1982, 218: 443-448.
[2]    Flowers T J, Yeo A R. Breeding for salinity resistance in crop plants: Where next Australian. Australian Journal Plant Physiology, 1995, 22: 875-884.
[3]    Nelson D E, Shen B, Bohnert H J. Salinity tolerance- mechanisms, models and the metabolic engineering of complex traits. Genetic Engineering, 1998, 20: 153-176.
[4]    郭三堆, 倪万潮, 徐琼芳. 编码杀虫蛋白融合基因和表达载体及其应用: 中国, ZL95119563.8.
Guo S D, Ni W C, Xu Q F. Encoding insecticidal protein fusion gene and expression vectors and its application: China, ZL95119563.8. (in Chinese)
[5]    Yu L H, Wu S J, Peng Y S,PENG Y S, LIU R N, CHEN X, ZHAO P, XU P, ZHU J B, JIAO G L, PEI Y, XIANG C B. Arabidopsis EDT1/HDG11 improves drought and salt tolerance in cotton and poplar and increases cotton yield in the field. Plant Biotechnology Journal, 2016, 14: 72-84.
[6]    张慧军, 董合忠, 石跃进, 陈受宜, 朱永红. 山菠菜胆碱单加氧酶基因对棉花的遗传转化和耐盐性表达. 作物学报, 2007, 33(7): 1073-1078.
Zhang H J, Dong H Z, Shi Y J, CHEN S Y, ZHU Y H. Transformation of cotton (Gossypium hirsutum L.) with AhCMO gene and the expression of salinity tolerance. Acta Agronomica Sinica, 2007, 33(7): 1073-1078. (in Chinese)
[7]    Pasapula V, Shen G X, Kuppu S, VALENCIA J P, MENDOZA M, HOU P, CHEN J, QIU X Y, ZHU L F, ZHANG X L, AULD D, BLUMWALD M, ZHANG H, GAXIOLA R, PAYTON P. Expression of an Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions. Plant Biotechnology Journal, 2011, 9: 88-99.
[8]    Serrano R, Gaxiola R. Microbial models and salt stress tolerance in plants. Critical Reviews in Plant Sciences, 1994, 13: 121-138.
[9]   Gaxiola R, de Larrinoa I F, Villalba J M, Serrano R. A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast. The EMBO Journal, 1992,11: 3157-3164.
[10]   RIOS G, FERRANDO A, SERRANO R. Mechanisms of salt tolerance conferred by overexpression of the HAL1 gene in Saccharomyces cerevisiae. Yeast, 1994,13: 515-528.
[11]   PARK J M, PARK C J, LEE S B, HAM B K, SHIN R, PAEK K H. Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2- type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco. The Plant Cell, 2001, 13: 1035-1046.
[12]   WINICOV I. Alfin1 transcription factor overexpression enhances plant root growth under normal and saline conditions and improves salt tolerance in alfalfa. Planta, 2000, 210: 416-422.
[13]   WINICOV I, BASTOLA D R. Transgenic overexpression of the transcription factor Alfin1 enhances expression of the endogenous MsPRP2 gene in alfalfa and improves salinity tolerance of the plants. Plant Physiology, 1999, 120: 473-480.
[14]   XU D P, DUAN X L, WANG B Y, HO T H, WU R. Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiology, 1996, 110: 249-257.
[15]   BORDAS M, MONTESINOS C, DABAUZA M, SALVADOR A, ROIG L A,SERRANO R, MORENO V. Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt tolerance. Transgenic Research, 1997, 6: 41-50.
[16]   GISBERT C, RUS A M, BOLARIN M C, CORONADO J.M L, ARRILLAGA I, MONTESINOS C, CARO M, SERRANO R, MORENO V. The yeast HAL1 gene improves salt tolerance of transgenic tomato. Plant Physiology, 2000, 123: 393-402.
[17]   Yang S X, Zhao Y X, Zhang Q, HE Y K, ZHANG H, LUO D. HAL1 mediate salt adaptation in Arabidopsis thaliana. Cell Research, 2001, 11: 142-148.
[18]   Ellul P, RIOS G, ATARES A, ROIG L A, SERRANO R, MORENO V. The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun & Nakai]. Theoretical and Applied Genetics, 2003, 107: 462-469.
[19]   刘艳芝, 韦正乙, 邢少辰, 谭化. 高明, 董英山. HAL1基因转化苜蓿再生植株及其耐盐性. 吉林农业科学, 2008, 33: 21-24.
LIU Y Z, WEI Z Y, XING S C, TAN H, GAO M, DONG Y S. Transgenetic alfalfa with HAL1 gene and its salt tolerance. Journal of Jilin Agricultural Sciences, 2008, 33: 21-24. (in Chinese)
[20]   Rus A M, Estan M T, Gisbert C, sogo b g, serrano r, caro m, moreno m, bolarin m c. Expressing the yeast HAL1 gene in tomato increases fruit yield and enhances K+/Na+ selectivity under salt stress. Plant Cell and Environment, 2001, 24: 875-880.
[21]   于志晶, 王岭, 金永梅, 林秀峰, 马瑞. 酵母HAL1基因转化水稻及其耐碱性研究. 吉林农业科学, 2014, 39: 17-20.
Yu Z J, Wang L, Jin Y M, LIN X F, MA R. Studies on transgenic rice with HAL1 gene and its tolerance to alkali. Journal of Jilin Agricultural Sciences, 2014, 39: 17-20. (in Chinese)
[22]   孔静静. 藻类耐盐相关基因的克隆及转化棉花的初步研究[D]. 开封: 河南大学, 2013.
Kong J J.Gene cloning of salt tolerance on algae and its preliminary research of transformation on cotton [D]. Kaifeng: Henan University, 2013. (in Chinese)
[23]   周凯. 陆地棉耐盐相关基因(GhPTACGhGnT)克隆及其表达分析[D]. 北京: 中国农业科学院, 2011.
Zhou K. Cloning and expression analysis of salt-tolerance related genes (GhPTACGhGnT) on Gossypium hirsutum L.[D]. Beijing: Chinese Academy of Agrecultural Sciences, 2011. (in Chinese)
[24]   张莹. 转TsVP-AtNHX1基因棉花的耐盐性研究[D]. 济南: 山东大学, 2014.
Zhang Y. The study of salt tolerance in cotton co-expressing TsVP and AtNHX1[D]. jinan: Shandong University, 2014. (in Chinese)
[25]   Yan J, Song W N, Eviatar N. A MAPK gene from Dead Sea fungus confers stress tolerance to lithium salt and freezing-thawing: Prospects for saline agriculture. Proceedings of the National Academy of Sciences of the USA, 2005, 102: 18992-18997.
[26]   Dietmar K. Evolution of the cellular stress proteome: from monophyletic origin to ubiquitous function. The Journal of Experimental Biology, 2003, 206: 3119-3124.
[27]   Zhao F Y, Guo S L, Zhang H, ZHAO Y X.Expression of yeast SOD2 in transgenic rice results in increased salt tolerance. Plant Science, 2006, 170: 216-224.
[28]   张丽青. 嗜热毛壳菌超氧化物歧化酶基因的克隆、表达及转基因烟草耐盐性研究[D]. 泰安: 山东农业大学, 2011.
Zhang L Q. Cloning, expression of Superoxide Dismutase (SOD) gene on Chaetomium thermophilum CT2 and salt resistance of transgenic tobacco[D]. Taian: Shandong agricultural university, 2011. (in Chinese)
[29]   谢丽霞. 极端嗜盐曲霉核糖体蛋白基因SpRPS3aeSpRPL44的克隆及其抗逆功能研究[D]. 长春: 吉林农业大学, 2013.
Xie L X. Cloning and stress resistance analys of ribosomal protein genes (SpRPS3ae and SpRPL44 ) in extreme halotolerant Aspergillus sp[D]. Changchun: Jilin agricultural university, 2013. (in Chinese)
[30]   张荃, 王淑芳, 赵彦修, 赵可夫, 张慧. HAL1基因转化番茄及耐盐转基因番茄的鉴定. 生物工程学报, 2001, 17(6): 659-662.
ZHANG Q, WANG S F, ZHAO Y X, ZHAO K F, ZHANG H. HAL1 gene transform into tomatoe and salt-tolerance identification of transgenic tomato. Chinese Journal of Biotechnology, 2001, 17(6): 659-662. (in Chinese)
[1] WANG CaiXiang,YUAN WenMin,LIU JuanJuan,XIE XiaoYu,MA Qi,JU JiSheng,CHEN Da,WANG Ning,FENG KeYun,SU JunJi. Comprehensive Evaluation and Breeding Evolution of Early Maturing Upland Cotton Varieties in the Northwest Inland of China [J]. Scientia Agricultura Sinica, 2023, 56(1): 1-16.
[2] LI YuZe,ZHU JiaWei,LIN Wei,LAN MoYing,XIA LiMing,ZHANG YiLi,LUO Cong,HUANG Gui Xiang,HE XinHua. Cloning and Interaction Protein Screening of RHF2A Gene from Xiangshui Lemon [J]. Scientia Agricultura Sinica, 2022, 55(24): 4912-4926.
[3] XIE XiaoYu, WANG KaiHong, QIN XiaoXiao, WANG CaiXiang, SHI ChunHui, NING XinZhu, YANG YongLin, QIN JiangHong, LI ChaoZhou, MA Qi, SU JunJi. Restricted Two-Stage Multi-Locus Genome-Wide Association Analysis and Candidate Gene Prediction of Boll Opening Rate in Upland Cotton [J]. Scientia Agricultura Sinica, 2022, 55(2): 248-264.
[4] WANG Juan, MA XiaoMei, ZHOU XiaoFeng, WANG Xin, TIAN Qin, LI ChengQi, DONG ChengGuang. Genome-Wide Association Study of Yield Component Traits in Upland Cotton (Gossypium hirsutum L.) [J]. Scientia Agricultura Sinica, 2022, 55(12): 2265-2277.
[5] QIN HongDe, FENG ChangHui, ZHANG YouChang, BIE Shu, ZHANG JiaoHai, XIA SongBo, WANG XiaoGang, WANG QiongShan, LAN JiaYang, CHEN QuanQiu, JIAO ChunHai. F1 Performance Prediction of Upland Cotton Based on Partial NCII Design [J]. Scientia Agricultura Sinica, 2021, 54(8): 1590-1598.
[6] LI TianCong,ZHU Hang,WEI Ning,LONG Feng,WU JianYing,ZHANG Yan,DONG JinGao,SHEN Shen,HAO ZhiMin. The Expression Pattern and Interaction Analysis of the Homologues of Splicing Factor SC35 in Setosphaeria turcica [J]. Scientia Agricultura Sinica, 2021, 54(4): 733-743.
[7] WANG Na,ZHAO ZiBo,GAO Qiong,HE ShouPu,MA ChenHui,PENG Zhen,DU XiongMing. Cloning and Functional Analysis of Salt Stress Response Gene GhPEAMT1 in Upland Cotton [J]. Scientia Agricultura Sinica, 2021, 54(2): 248-260.
[8] SUN YuChen,JIA RuiPu,FAN KuoHai,SUN Na,SUN YaoGui,SUN PanPan,LI HongQuan,YIN Wei. Detection of Interaction Between Porcine Type I Complement Receptor and C3b Active Fragment in Vitro [J]. Scientia Agricultura Sinica, 2021, 54(19): 4243-4254.
[9] MA Lin,WEN HongYu,WANG XueMin,GAO HongWen,PANG YongZhen. Cloning and Function Analysis of MsMAX2 Gene in Alfalfa (Medicago sativa L.) [J]. Scientia Agricultura Sinica, 2021, 54(19): 4061-4069.
[10] WEI Xin, WANG HanTao, WEI HengLing, FU XiaoKang, MA Liang, LU JianHua, WANG XingFen, YU ShuXun. Cloning and Drought Resistance Analysis of GhWRKY33 in Upland Cotton [J]. Scientia Agricultura Sinica, 2020, 53(22): 4537-4549.
[11] QU YuJie, SUN JunLing, GENG XiaoLi, WANG Xiao, Zareen Sarfraz, JIA YinHua, PAN ZhaoE, HE ShouPu, GONG WenFang, WANG LiRu, PANG BaoYin, DU XiongMing. Correlation Between Genetic Distance of Parents and Heterosis in Upland Cotton [J]. Scientia Agricultura Sinica, 2019, 52(9): 1488-1501.
[12] Wei ZHANG,JinJun DAI,XueHai YANG,JinTao WEI,MingXin CHEN,JunPeng HU,ShaoWen HUANG. Evaluation of Apparent Metabolic Energy, Nitrogen Corrected Metabolic Energy, Biological Value of Protein and Ileal Digestibility of Amino Acid of Yeast Hydrolysate for Broilers [J]. Scientia Agricultura Sinica, 2019, 52(20): 3685-3694.
[13] CUI TianTian, YAN JianHong, BIN Yu, LI ZhongAn, ZHOU ChangYong, SONG Zhen. Construction of Citrus leaf blotch virus Infectious cDNA Clone by Yeast Homologous Recombination System [J]. Scientia Agricultura Sinica, 2018, 51(9): 1695-1705.
[14] ZHANG HuiYuan,LIU YongWei,YANG JunFeng,ZHANG ShuangXi,YU TaiFei,CHEN Jun,CHEN Ming,ZHOU YongBin,MA YouZhi,XU ZhaoShi,FU JinDong. Identification and Analysis of Salt Tolerance of Wheat Transcription Factor TaWRKY33 Protein [J]. Scientia Agricultura Sinica, 2018, 51(24): 4591-4602.
[15] XU HaiFeng,YANG GuanXian,WANG YiCheng,JIANG ShengHui,WANG Nan,CHEN XueSen. Apple MdMYB32 Inhibits the Anthocyanin Biosynthesis by Its Own EAR Inhibitory Sequence [J]. Scientia Agricultura Sinica, 2018, 51(24): 4690-4699.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!