Effect of overexpression of the alfalfa MsHB7 gene on drought tolerance of Arabidopsis

doi:10.11686/cyxb2020181

Abstract

Abstract:

The homeodomain-leucine zipper （HD-Zip） protein subfamily I plays an important role in the regulation of plant abiotic stress. There are few reports of HD-Zip I genes in alfalfa， although they have been cloned and identified in many species. The purpose of this study was to investigate the role of MsHB7， a HD-Zip I gene from alfalfa， in the regulation of Arabidopsis thaliana drought resistance. The MsHB7 gene with an open reading frame of 738 bp and encoding 245 amino acids was obtained by cloning. Multiple sequence alignment and phylogenetic tree analysis showed that MsHB7 belonged to the HD-Zip I subfamily and was closely related to ATHB7 and ATHB12 in A. thaliana. Real-time fluorescence quantitative analysis showed that the MsHB7 gene was induced by drought. The MsHB7 gene was transformed into A. thaliana and positive plants were obtained. After drought treatment， the transgenic A. thaliana wilted more obviously. The relative water content of transgenic plants was significantly lower than that of wild-type A. thaliana， and the proline and malondialdehyde levels accumulated in transgenic plants were more than wild-type. qRT-PCR results showed that the expression levels of stress response genes ATCAT1， ATDREB2A and ATRD29A in the transgenic A. thaliana were significantly increased， while the expression levels of ATLEA3 were significantly decreased after stress. The above results indicate that the overexpression of MsHB7 gene can reduce the drought tolerance of transgenic A. thaliana， which provides theoretical information as a basis for the further development and utilization of this gene.

Key words: alfalfa, homeodomain-leucine zipper, drought stress, transgenic Arabidopsis

Yi-yao HOU, Xiao LI, Rui-cai LONG, Qing-chuan YANG, Jun-mei KANG, Chang-hong GUO. Effect of overexpression of the alfalfa MsHB7 gene on drought tolerance of Arabidopsis[J]. Acta Prataculturae Sinica, 2021, 30(4): 170-179.

Figures/Tables 9

References 33

1	Liu D， Yang L， Luo M， et al. Molecular cloning and characterization of PtrZPT2-1， a ZPT2 family gene encoding a Cys2/His2-type zinc finger protein from trifoliate orange （Poncirus trifoliata L. Raf.） that enhances plant tolerance to multiple abiotic stresses. Plant Science， 2017， 263： 66-78.
2	Nakashima K， Ito Y， Yamaguchi-shinozaki K. Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiology， 2009， 149（1）： 88-95.
3	Tang L L， Cai H， Ji W， et al. Overexpression of GsZFP1 enhances salt and drought tolerance in transgenic alfalfa （Medicago sativa L.）. Plant Physiology and Biochemistry， 2013， 71： 22-30.
4	Duan J Z， Li Y， Zhao M Z， et al. Progress on application of NAC transcription factors in plant abiotic tolerance genetic engineering. Crops， 2017（2）： 14-22.
	段俊枝，李莹，赵明忠，等. NAC转录因子在植物抗非生物胁迫基因工程中的应用进展. 作物杂志， 2017（2）： 14-22.
5	Ariel F D， Manavella P A， Dezar C A， et al. The true story of the HD-Zip family. Trends in Plant Science， 2007， 12（9）： 419-426.
6	Vollbrecht E， Veit B， Sinha N， et al. The developmental gene Knotted-1 is a member of a maize homeobox gene family. Nature， 1991， 350： 241-243.
7	Harris J C， Hrmova M， Lopato S， et al. Modulation of plant growth by HD-Zip class I and Ⅱ transcription factors in response to environmental stimuli. New Phytologist， 2011， 190（4）： 823-837.
8	Zhao Y， Zhou Y Q， Jiang H Y， et al. Systematic analysis of sequences and expression patterns of drought-responsive members of the HD-Zip gene family in maize. PLoS One， 2011， 6（12）： e28488.
9	Söderman E， Hjellström M， Fahleson J， et al. The HD-Zip gene ATHB6 in Arabidopsis is expressed in developing leaves， roots and carpels and up-regulated by water deficit conditions. Plant Molecular Biology， 1999， 40（6）： 1073-1083.
10	Söderman E， Mattsson J， Engström P. The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid. the Plant Journal， 1996， 10（2）： 375-381.
11	Olsson A S B， Engström P， Söderman E. The homeobox genes ATHB12 and ATHB7 encode potential regulators of growth in response to water deficit in Arabidopsis. Plant Molecular Biology， 2004， 55（5）： 663-677.
12	Hjellström M， Olsson A S B， Engström P， et al. Constitutive expression of the water deficit-inducible homeobox gene ATHB7 in transgenic Arabidopsis causes a suppression of stem elongation growth. Plant， Cell & Environment， 2003， 26（7）： 1127-1136.
13	Turchi L， Carabelli M， Ruzza V， et al. Arabidopsis HD-Zip Ⅱ transcription factors control apical embryo development and meristem function. Development， 2013， 140（10）： 2118-2129.
14	Prigge M J， Otsuga D， Alonso J M， et al. Class Ⅲ homeodomain-leucine zipper gene family members have overlapping， antagonistic， and distinct roles in Arabidopsis development. the Plant Cell， 2005， 17（1）： 61-76.
15	Rerie W G， Feldmann K A， Marks M D. The GLABRA2 gene encodes a homeo domain protein required for normal trichome development in Arabidopsis. Genes & Development， 1994， 8（12）： 1388-1399.
16	Zhao M R， Shen Y H， Li Y C， et al. Research progress in the genetic engineer of alfalfa stress resistance. Acta Agrestia Sinica， 2014， 22（2）： 243-248.
	赵美荣，申玉华，李永春，等. 紫花苜蓿抗逆基因工程研究进展. 草地学报， 2014， 22（2）： 243-248.
17	Kumar S， Stecher G， Tamura K. MEGA7： Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and Evolution， 2016， 33（7）： 1870-1874.
18	Li Z Y. Molecular cloning and functional analysis of MsHSP70 gene in Medicago sativa L. Beijing： Chinese Academy of Agricultural Science， 2015.
	栗振义. 紫花苜蓿热激蛋白基因MsHSP70的克隆及功能分析. 北京：中国农业科学院， 2015.
19	Roodbarkelari F， Groot E P. Regulatory function of homeodomain-leucine zipper （HD-ZIP） family proteins during embryogenesis. New Phytologist， 2017， 213（1）： 95-104.
20	Sen S， Chakraborty J， Ghosh P， et al. Chickpea WRKY70 regulates the expression of a Homeodomain-Leucine Zipper （HD-Zip） I transcription factor CaHDZ12， which confers abiotic stress tolerance in transgenic tobacco and chickpea. Plant & Cell Physiology， 2017， 58（11）： 1934-1952.
21	Zuo Z F， Kang H G， Park M Y， et al. Zoysia japonica MYC type transcription factor ZjICE1 regulates cold tolerance in transgenic Arabidopsis. Plant Science， 2019， 289： 110254.
22	Li Q， Wu Q， Wang A， et al. Tartary buckwheat transcription factor FtbZIP83 improves the drought/salt tolerance of Arabidopsis via an ABA-mediated pathway. Plant Physiology and Biochemistry， 2019， 144： 312-323.
23	Ju Y L， Yue X F， Min Z， et al. VvNAC17， a novel stress-responsive grapevine （Vitis vinifera L.） NAC transcription factor， increases sensitivity to abscisic acid and enhances salinity， freezing， and drought tolerance in transgenic Arabidopsis. Plant Physiology and Biochemistry， 2020， 146： 98-111.
24	Zhao Y， Ma Q， Jin X L， et al. A novel maize homeodomain-leucine zipper （HD-Zip） I gene， Zmhdz10， positively regulates drought and salt tolerance in both rice and Arabidopsis. Plant & Cell Physiology， 2014， 55（6）： 1142-1156.
25	Tang Y H， Bao X X， Wang S， et al. A Physic nut stress-responsive HD-Zip transcription factor， JcHDZ07， confers enhanced sensitivity to salinity stress in transgenic Arabidopsis. Frontiers in Plant Science， 2019， 10： 942.
26	Zhang S X， Haider I， Kohlen W， et al. Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Molecular Biology， 2012， 80（6）： 571-585.
27	Li M N， Long R C， Yang Q C， et al. Cloning and function analysis of a salt-stress-induced HD-Zip transcription factor MsHB2 from alfalfa. Scientia Agricultura Sinica， 2014， 47（4）： 622-632.
	李明娜，龙瑞才，杨青川，等. 紫花苜蓿盐诱导HD-Zip类转录因子MsHB2的克隆及功能分析. 中国农业科学， 2014， 47（4）： 622-632.
28	Zhang H， Wang P H. Determination of relative water content in plant leaves in vivo. Plant Physiology Communications， 1991（3）： 217-219.
	张慧，汪沛洪. 叶片相对含水量的活体测定. 植物生理学报， 1991（3）： 217-219.
29	Kong W W， Liu F， Zhang C， et al. Non-destructive determination of malondialdehyde （MDA） distribution in oilseed rape leaves by laboratory scale NIR hyperspectral imaging. Scientific Reports， 2016， 6（1）： 35393.
30	Székely G， Abrahám E， Cséplo A， et al. Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. the Plant Journal， 2008， 53（1）： 11-28.
31	Jiang H Y， Teng K， Tan P H， et al. Heterogeneous expression of a novel Zoysia japonica C₂H₂ zinc finger protein gene， ZjZFN1， caused drought sensitivity in Arabidopsis. Acta Prataculturae Sinica， 2019， 28（4）： 129-138.
	姜红岩，滕珂，檀鹏辉，等. 日本结缕草 ZjZFN1 基因对拟南芥的转化及其耐旱性分析. 草业学报， 2019， 28（4）： 129-138.
32	Pruthvi V， Narasimhan R， Nataraja K N， et al. Simultaneous expression of abiotic stress responsive transcription factors， AtDREB2A， AtHB7 and AtABF3 improves salinity and drought tolerance in peanut （Arachis hypogaea L.）. PLoS One， 9（12）： e111152.
33	Qin F， Kakimoto M， Sakuma Y， et al. Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L. the Plant Journal， 2007， 50（1）： 54-69.

引物Primer	引物Sequence (5'-3')
MsHB7F	CAAAACTTAGGCCTTAGCCATATAT
MsHB7R	GCAACATAGAAGAACATGGTGCA
qMsHB7F	ATGAGGGTTTGGAGGATAAAATCGT
qMsHB7R	CAAGTCCAAAAATCCAACCATTGAG
qMsactin2F	CAAAAGATGGCAGATGCTGAGGAT
qMsactin2R	CATGCACCAGTATGACGAGGTCG
pMsHB7F	TGCTCTAGAATGATGGAGGAAGAAGAG
pMsHB7R	ACGGGATCCTCAAGTCCAAAAATCC
MsHB7F1	CGACACACTTGTCTACTCCAAAAAT
MsHB7R1	TTCAAGTCCAAAAATCCAACCATTG
qMsHB7F1	TGGAGCCAAGGAAGAAGATGC
qMsHB7R1	CCATCATGCGATGTTTCCACC
qATactin7F	AGCTAGAGACAGCCAAGAGC
qATactin7R	GCTTCCATTCCGATGAGCGA
qATCAT1F	CGCCATGCCGAAAAATACCC
qATCAT1R	CTTGCCTGTCTGAATCCCAGGAC
qATDREB2AF	CTGGAGAATGGTGCGGAAGA
qATDREB2AR	CAGATAGCGAATCCTGCTGTTGT
qATLEA3F	GATTGACCCGGCTGAGCTACGA
qATLEA3R	AGATGGGATTCACCACAAAAGA
qATRD29AF	GATATCGACAAGGATGTGCCG
qATRD29AR	GTATCCAGGTCTTCCCTTCGC

引物Primer	引物Sequence (5'-3')
MsHB7F	CAAAACTTAGGCCTTAGCCATATAT
MsHB7R	GCAACATAGAAGAACATGGTGCA
qMsHB7F	ATGAGGGTTTGGAGGATAAAATCGT
qMsHB7R	CAAGTCCAAAAATCCAACCATTGAG
qMsactin2F	CAAAAGATGGCAGATGCTGAGGAT
qMsactin2R	CATGCACCAGTATGACGAGGTCG
pMsHB7F	TGCTCTAGAATGATGGAGGAAGAAGAG
pMsHB7R	ACGGGATCCTCAAGTCCAAAAATCC
MsHB7F1	CGACACACTTGTCTACTCCAAAAAT
MsHB7R1	TTCAAGTCCAAAAATCCAACCATTG
qMsHB7F1	TGGAGCCAAGGAAGAAGATGC
qMsHB7R1	CCATCATGCGATGTTTCCACC
qATactin7F	AGCTAGAGACAGCCAAGAGC
qATactin7R	GCTTCCATTCCGATGAGCGA
qATCAT1F	CGCCATGCCGAAAAATACCC
qATCAT1R	CTTGCCTGTCTGAATCCCAGGAC
qATDREB2AF	CTGGAGAATGGTGCGGAAGA
qATDREB2AR	CAGATAGCGAATCCTGCTGTTGT
qATLEA3F	GATTGACCCGGCTGAGCTACGA
qATLEA3R	AGATGGGATTCACCACAAAAGA
qATRD29AF	GATATCGACAAGGATGTGCCG
qATRD29AR	GTATCCAGGTCTTCCCTTCGC

[1]	Di ZHANG, Li-fei REN, Guang-bin LIU, Fu-qing LUO, Wen-hao ZHANG, Tian-zuo WANG. Comparative metabolite profiling of alfalfa seeds dried at different temperatures [J]. Acta Prataculturae Sinica, 2021, 30(3): 158-166.
[2]	Kai-qiang LIU, Wen-hui LIU, Zhi-feng JIA, Guo-ling LIANG, Xiang MA. Effects of drought stress on yield and dry matter accumulation and distribution of Avena sativa cv. Qingyan No.1 [J]. Acta Prataculturae Sinica, 2021, 30(3): 177-188.
[3]	Bai-ping SHA, Ying-zhong XIE, Xue-qin GAO, Wei CAI, Bing-zhe FU. Effects of coupling of drip irrigation water and fertilizer on yield and quality of alfalfa in the yellow river irrigation district [J]. Acta Prataculturae Sinica, 2021, 30(2): 102-114.
[4]	Shuang LIU, Fu-ping HUI. Distribution of alfalfa in the Ming and Qing Dynasties and the underlying driving factors [J]. Acta Prataculturae Sinica, 2021, 30(2): 178-189.
[5]	Dong LI, Hong-tao SHEN, Yan-fang WANG, Yue-hua WANG, Li-jun WANG, Shi-min ZHAO, Ling LIU. Effects of exogenous melatonin on photosynthetic carbon assimilation and endogenous hormones in tobacco seedlings under drought stress [J]. Acta Prataculturae Sinica, 2021, 30(1): 130-139.
[6]	Zhen-song LI, Li-qiang WAN, Shuo LI, Xiang-lin LI. Response of alfalfa root architecture and physiological characteristics to drought and rehydration [J]. Acta Prataculturae Sinica, 2021, 30(1): 189-196.
[7]	WU Yong, LIU Xiao-jing, LIN Fang, TONG Chang-chun. A data envelopment analysis study of alfalfa fertilization responses and economic return in the desert irrigation area of Hexi [J]. Acta Prataculturae Sinica, 2020, 29(9): 94-105.
[8]	XING Yi-mei, DONG Li, ZHAN Li-feng, CAI Hua, YANG Sheng-qiu, SUN Na. Effect of mixed inoculation of Glomus mosseae and Sinorhizobium melilotion alkali resistance of alfalfa [J]. Acta Prataculturae Sinica, 2020, 29(9): 136-145.
[9]	QIN Feng-fei, LI Zhi-hua, LIU Xin-bao, QU Hui, PINGCUO Zhuo-ma, LUOSONG Qun-cuo, SU Meng-han. Effects of exogenous 2, 4-epibrassinolide on the growth and photosynthesis of alfalfa under high temperature and low light stress in summer [J]. Acta Prataculturae Sinica, 2020, 29(9): 146-160.
[10]	TONG Chang-chun, LIU Xiao-jing, LIN Fang, YU Tie-feng. Yield effect of optimisation of photosynthetic characteristics of alfalfa through balanced fertilization [J]. Acta Prataculturae Sinica, 2020, 29(8): 70-80.
[11]	LU Jiao-yun, XIONG Jun-bo, ZHANG He-shan, TIAN Hong, YANG Hui-min, LIU Yang. Effects of water stress on yield, quality and trace element composition of alfalfa [J]. Acta Prataculturae Sinica, 2020, 29(8): 126-133.
[12]	ZENG Ling-shuang, LI Pei-ying, SUN Xiao-fan, SUN Zong-jiu. A multi-trait evaluation of drought resistance of bermudagrass (Cynodon dactylon) germplasm from different habitats in Xinjiang province [J]. Acta Prataculturae Sinica, 2020, 29(8): 155-169.
[13]	ZHANG Yu-jun, SHANG Yi-shun, WANG Pu-chang, DING Lei-lei, ZHANG Wen, ZOU Chao. Effects of super absorbent polymers on growth and physiological characteristics of Sophora davidii vs. Panjiang seedlings under drought stress [J]. Acta Prataculturae Sinica, 2020, 29(7): 90-98.
[14]	CAI Lu, WANG Lin-lin, LUO Zhu-zhu, LI Ling-ling, NIU Yi-ning, CAI Li-qun, XIE Jun-hong. Meta-analysis of alfalfa yield and WUE response to growing ages in China [J]. Acta Prataculturae Sinica, 2020, 29(6): 27-38.
[15]	ZHANG Li-li, SHI Min, LI Yan-zhong. Effect of anthracnose infection on alfalfa yield and quality in the Shaerqin area [J]. Acta Prataculturae Sinica, 2020, 29(6): 117-126.