The Plantlets of Medicago polymorpha L. and Medicago sativa L. Under Salt Stress: Transcriptome Analysis and Salt Tolerance Gene Screening

doi:10.11924/j.issn.1000-6850.casb2022-0235

Abstract

Abstract:

In order to screen the salt tolerance genes of Medicago polymorpha L. and Medicago sativa L., the plantlets of Medicago polymorpha L. (JHC) and Medicago sativa L. (ZHMX) under salt stress were used for transcriptome analysis. The results showed that the number of differential genes between JHC group and ZHMX group was 26722. 15850 genes were down regulated and 10872 genes were up regulated in JHC group vs ZHMX group. Pathways with significant enrichment of KEGG gene in JHC group and ZHMX group included ribosome, photosynthesis antenna protein, glycolysis/gluconeogenesis, photosynthesis, phenylpropanoid biosynthesis, etc. Salt tolerance genes in JHC group included basic leucine zipper 43, NAC transcription factor 47, ABC transporter A family member 7, late embryogenesis abundant protein 2, ethylene responsive transcription factor ERF110, etc. The salt tolerance genes in ZHMX group included WRKY transcription factor, protein TIFY 8, transcription factor MYB13, nuclear transport factor 2A, low temperature and salt responsive protein, etc. The results of this experiment could provide a theoretical basis for understanding the molecular mechanism of salt tolerance of Medicago polymorpha L. and Medicago sativa L. and breeding new salt tolerant varieties of Medicago polymorpha L. and Medicago sativa L..

Key words: salt stress, Medicago polymorpha L., Medicago sativa L., plantlet, transcriptome analysis, salt tolerance gene

HONG Senrong, ZHU Yingying, LI Ziying, HU Mingyan, OUYANG Kehui. The Plantlets of Medicago polymorpha L. and Medicago sativa L. Under Salt Stress: Transcriptome Analysis and Salt Tolerance Gene Screening[J]. Chinese Agricultural Science Bulletin, 2023, 39(3): 111-118.

Figures/Tables 6

References 38

[1]	江舟, 陈丰, 王东军, 等. 金花菜与燕麦间作对牧草产量与品质的影响[J]. 中国草地学报, 2020, 42(5):127-135.
[2]	任海龙, 魏臻武, 乔志宏, 等. 温度和硬实对金花菜种子萌发的影响[J]. 中国野生植物资源, 2019, 38(6):49-52.
[3]	CHARMAN N, BALLARD R A. Burr medic (Medicago polymorpha L.) selections for improved N2 fixation with naturalised soil rhizobia[J]. Soil biology & biochemistry, 2004, 36(8):1331-1337. doi: 10.1016/j.soilbio.2004.04.014 URL
[4]	ALTINOK S, YURTSEVEN E, AVCI S, et al. The effects of different irrigation water salinities and leaching ratios on green and dry forage yields of alfalfa (Medicago sativa L.)[J]. Agriculture & forestry, 2015, 61(1): 85-90.
[5]	CHEN Z, WANG Q Z. Graphene ameliorates saline-alkaline stress-induced damage and improves growth and tolerance in alfalfa (Medicago sativa L.)[J]. Plant physiology and biochemistry, 2021, 163: 128-138. doi: 10.1016/j.plaphy.2021.03.039 URL
[6]	CAMPBELL M T, KNECHT A, BERGER B, et al. Integrating image-based phenomics and association analysis to dissect the genetic architecture of temporal salinity responses in rice[J]. Plant physiology, 2015, 168(4):1476-1697. doi: 10.1104/pp.15.00450 pmid: 26111541
[7]	施田野, 顾宇蓝, 张磊, 等. 粗山羊草响应盐胁迫转录组分析[J]. 分子植物育种, 2020, 18(21):7015-7022.
[8]	赵嫚, 陈仕勇, 李亚萍, 等. 外源GABA对盐胁迫下金花菜种子萌发及幼苗抗氧化能力的影响[J]. 江苏农业学报, 2021, 37(2):310-316.
[9]	郭湘, 郭一帆, 黄思怡, 等. 干旱和盐胁迫对14个紫花苜蓿品种种子萌发特性的影响[J]. 草业科学, 2019, 36(9):2292-2303.
[10]	曹璇, 郑晓冬. 盐胁迫培养下季也蒙毕赤酵母的转录组学差异分析[J]. 浙江大学学报:农业与生命科学版, 2020, 46(4):400-406.
[11]	朱晓林, 魏小红, 王宝强, 等. c-GMP诱导对盐胁迫下番茄的转录组分析[J]. 浙江农业学报, 2020, 32(10):1788-1797. doi: 10.3969/j.issn.1004-1524.2020.10.07
[12]	卢锐, 李培英. 偃麦草盐胁迫下转录组分析[J]. 草地学报, 2020, 28(1):31-44. doi: 10.11733/j.issn.1007-0435.2020.01.004
[13]	马进, 郑钢. 利用转录组测序技术鉴定紫花苜蓿根系盐胁迫应答基因[J]. 核农学报, 2016, 30(8):1470-1479. doi: 10.11869/j.issn.100-8551.2016.08.1470
[14]	李红, 李波, 邬婷婷, 等. 紫花苜蓿耐苏打盐碱相关基因的转录组学分析[J]. 草地学报, 2019, 27(4):848-858. doi: 10.11733/j.issn.1007-0435.2019.04.008
[15]	屈兴红, 曾洪学. 金冕草(Paspalum notatum)盐胁迫转录组差异性分析[J]. 分子植物育种, 2019, 17(11):3515-3523.
[16]	热依麦阿依·阿布都艾尼, 陈静, 陈芸, 等. 盐胁迫下棉花根系的转录组分析及耐盐基因筛选[J]. 华南师范大学学报:自然科学版, 2020, 52(5):85-92.
[17]	ZOU C, JIANG W, YU D. Male gametophyte-specific WRKY34 transcription factor mediates cold sensitivity of mature pollen in Arabidopsis[J]. Journal of experimental botany, 2010, 61(14):3901-3914. doi: 10.1093/jxb/erq204 URL
[18]	EULGEM T, RUSHTON P J, ROBATZEK S, et al. The WRKY superfamily of plant transcription factors[J]. Trend in plant science, 2000, 5(5):199-206. doi: 10.1016/S1360-1385(00)01600-9 URL
[19]	王如月, 文武武, 赵恩华, 等. 紫花苜蓿MsWRKY11基因的克隆及其耐盐功能分析[J]. 草业学报, 2021, 30(11):157-169.
[20]	裴翠明, 张振亚, 马进. 南方型紫花苜蓿叶片盐胁迫应答转录因子鉴定与分析[J]. 浙江农业学报, 2016, 28(4):550-557.
[21]	马进, 郑钢, 裴翠明, 等. 南方型紫花苜蓿根系盐胁迫应答转录因子鉴定与分析[J]. 浙江农林大学学报, 2016, 33(2):201-208.
[22]	马进, 郑钢. 南方型紫花苜蓿叶片盐胁迫应答基因鉴定与分析[J]. 农业生物技术学报, 2015, 23(12):1531-1541.
[23]	ZHU D, BAI X, LUO X, et al. Identification of wild soybean (Glycine soja) TIFY family genes and their expression profiling analysis under bicarbonate stress[J]. Plant cell reports, 2013, 32(2):263-272. doi: 10.1007/s00299-012-1360-7 pmid: 23090726
[24]	侯相寅, 张文凯, 喻金秋, 等. 紫花苜蓿(Medicago sativa L.)ify基因家族的鉴定、生物信息学及表达模式分析[J]. 分子植物育种.
[25]	TAKAIWA F. A rice transmembrane bZIP transcription factor, OsbZIP39, regulates the endoplasmic reticulum stress response[J]. Plant & cell physiology, 2012, 53(1):144-53
[26]	曹晓宇, 王学敏, 郭继承, 等. 紫花苜蓿ZIP1转录因子MsbZIP1的克隆、表达特性和遗传转化分析[J]. 草地学报, 2020, 28(3):613-622. doi: 10.11733/j.issn.1007-0435.2020.03.004
[27]	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[J]. 中国农业科学, 2014, 47(4):622-632.
[28]	杨晶婷, 余艳, 高晓蓉, 等. MYB类转录因子调控植物耐逆机制的研究进展[J]. 杭州师范大学学报:自然科学版, 2021, 20(6):621-627.
[29]	LIU X, HONG L, LI X Y, et al. Improved drought and salt tolerance in transgenic Arabidopsis overexpressing a NAC transcriptional factor from Arachis hypogaea[J]. Journal of the agricultural chemical society of Japan, 2011, 75(3):443-450.
[30]	申玉华, 徐振军, 杨晓坡, 等. 紫花苜蓿NAC转录因子MsNAC1基因的克隆、生物信息学分析及非生物逆境胁迫下的表达分析[J]. 植物遗传资源学报, 2014, 15(6):1312-1319.
[31]	宋玉光, 张召然, 吕娇, 等. 紫苜蓿盐诱导NAC转录因子的鉴定、克隆及表达分析[J]. 植物生理学报, 2020, 56(7):1493-1503.
[32]	邵若玄, 沈忆珂, 周文彬, 等. 植物 ATP 结合盒(ABC)转运蛋白研究进展[J]. 浙江农林大学学报, 2013, 30(5):761-768.
[33]	CHEN K, SU C, TANG W, et al. Nuclear transport factor GmNTF2B enhances soybean drought tolerance by interacting with oxidoreductase GmOXR17 to reduce ROS content[J]. The plant journal, 2021, 107(3):15319.
[34]	刘晓敏, 张莉弘, 刘金亮, 等. 玉米逆境诱导型启动子克隆及其植物表达载体构建[J]. 生物技术通报, 2011(3):86-90.
[35]	郑洁旋, 张会, 简曙光, 等. 厚藤胚胎发生晚期丰富蛋白基因IpLEA 的克隆与功能分析建[J]. 分子植物育种, 2018, 16(13):4235-4244.
[36]	贾会丽, 王学敏, 郭继承, 等. 紫花苜蓿MsLEA4-4基因的克隆、表达分析及遗传转化[J]. 草业学报, 2017, 26(12):108-116.
[37]	RIECHMANN J L, HEARD J, MARTIN G, et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[J]. Science, 2000, 290(5499):2105-2110. doi: 10.1126/science.290.5499.2105 URL
[38]	坚伟宁, 左朋, 张国珍, 等. 紫花苜蓿MsERF003的基因克隆及其在干旱胁迫中的功能分析[J]. 分子植物育种, 2020, 18(17):5674-5681.

目的基因	F	上游	R	下游	退火温度/℃	长度/bp
WRKY转录因子	G1-F	TAATGTTTAGGCCCCTTTTCC	G1-R	CCTTTTGATTATTTCCTATGCTGAT	48.9	194
蛋白TIFY 8	G2-F	GAAACAGGGTTAGTGAGTAATGT	G2-R	GTTGGAGTAGATACACGGTCA	50.8	113
碱性亮氨酸拉链43	G3-F	CTCCAAGACTTCAGTCCTCCAT	G3-R	CATTCCTTAGCCAAACCACCT	52.9	199
转录因子MYB13	G4-F	TCTGGAAGAATTTCATCAGGGTA	G4-R	GTCAGAAGCAGCAGCATTGGA	48.9	111
NAC转录因子47	G5-F	GCACCAGCTTTCAAACATAGA	G5-R	TGTGGTAGTTTGGATTAGCCT	48.6	183
ABC转运A家族成员7	G6-F	AAGCCAACCTCAGGTACAGCC	G6-R	TGTCAAGATTTCCCACAGCAA	51	117
核运输因子2A	G7-F	TTACTTCCCTTCCTTTCCAGC	G7-R	GTGTTGTTCACCAGCGAGTTG	53.4	125
低温盐敏蛋白	G8-F	TGATTCTCACCATTTTGGGCTAT	G8-R	TGACGCCCCTGACAACTATTC	50.5	135
晚期胚胎发生丰富蛋白2	G9-F	CTTAACTGCCTCAGTAGCACC	G9-R	TACCAAAGAAACTGCCCAATC	50	106
乙烯反应转录因子ERF110	G10-F	CCATAGGACAAGACATTGCCACCTC	G10-R	AAGGGATTCAGGAAAGTTCGTGAGG	54.5	128
甘油醛磷酸脱氢酶	GAPDH-F	TCACGCCATCACAGCCACTCA	GAPDH-R	AACATCGACGGTTGGGACACG	56.2	187

目的基因	F	上游	R	下游	退火温度/℃	长度/bp
WRKY转录因子	G1-F	TAATGTTTAGGCCCCTTTTCC	G1-R	CCTTTTGATTATTTCCTATGCTGAT	48.9	194
蛋白TIFY 8	G2-F	GAAACAGGGTTAGTGAGTAATGT	G2-R	GTTGGAGTAGATACACGGTCA	50.8	113
碱性亮氨酸拉链43	G3-F	CTCCAAGACTTCAGTCCTCCAT	G3-R	CATTCCTTAGCCAAACCACCT	52.9	199
转录因子MYB13	G4-F	TCTGGAAGAATTTCATCAGGGTA	G4-R	GTCAGAAGCAGCAGCATTGGA	48.9	111
NAC转录因子47	G5-F	GCACCAGCTTTCAAACATAGA	G5-R	TGTGGTAGTTTGGATTAGCCT	48.6	183
ABC转运A家族成员7	G6-F	AAGCCAACCTCAGGTACAGCC	G6-R	TGTCAAGATTTCCCACAGCAA	51	117
核运输因子2A	G7-F	TTACTTCCCTTCCTTTCCAGC	G7-R	GTGTTGTTCACCAGCGAGTTG	53.4	125
低温盐敏蛋白	G8-F	TGATTCTCACCATTTTGGGCTAT	G8-R	TGACGCCCCTGACAACTATTC	50.5	135
晚期胚胎发生丰富蛋白2	G9-F	CTTAACTGCCTCAGTAGCACC	G9-R	TACCAAAGAAACTGCCCAATC	50	106
乙烯反应转录因子ERF110	G10-F	CCATAGGACAAGACATTGCCACCTC	G10-R	AAGGGATTCAGGAAAGTTCGTGAGG	54.5	128
甘油醛磷酸脱氢酶	GAPDH-F	TCACGCCATCACAGCCACTCA	GAPDH-R	AACATCGACGGTTGGGACACG	56.2	187

目的基因	RNA-Seq		qRT-PCR
目的基因	金花菜	紫花苜蓿	金花菜	紫花苜蓿
WRKY转录因子	0.07	2.33	0.26 b	1 a
蛋白TIFY 8	0.59	3.70	0.21 b	1 a
碱性亮氨酸拉链43	54.78	10.56	5.04 a	1 b
转录因子MYB13	5.50	23.47	0.26 b	1 a
NAC转录因子47	237.73	71.87	1.93 a	1 b
ABC转运A家族成员7	48.55	4.99	2.29 a	1 b
核运输因子2A	13.08	136.74	0.28 b	1 a
低温盐响应蛋白	0.22	136.59	0.24 b	1 a
晚期胚胎发生丰富蛋白2	5080.76	0.89	1262.52 a	1 b
乙烯反应转录因子ERF110	176.64	16.00	8.92 a	1 b

目的基因	RNA-Seq		qRT-PCR
目的基因	金花菜	紫花苜蓿	金花菜	紫花苜蓿
WRKY转录因子	0.07	2.33	0.26 b	1 a
蛋白TIFY 8	0.59	3.70	0.21 b	1 a
碱性亮氨酸拉链43	54.78	10.56	5.04 a	1 b
转录因子MYB13	5.50	23.47	0.26 b	1 a
NAC转录因子47	237.73	71.87	1.93 a	1 b
ABC转运A家族成员7	48.55	4.99	2.29 a	1 b
核运输因子2A	13.08	136.74	0.28 b	1 a
低温盐响应蛋白	0.22	136.59	0.24 b	1 a
晚期胚胎发生丰富蛋白2	5080.76	0.89	1262.52 a	1 b
乙烯反应转录因子ERF110	176.64	16.00	8.92 a	1 b