Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (23): 4397-4408.doi: 10.3864/j.issn.0578-1752.2018.23.001

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

Isolation and Functional Analysis of Soybean GmLEA in Seed Vigor

ZHOU YaLi(),ZHU YaJing,ZHAO FeiYun,WANG Shuang,LIU SuShuang,GUO LingKai,ZHAO HaiHong,MA Hao()   

  1. National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095
  • Received:2018-06-22 Accepted:2018-07-29 Online:2018-12-01 Published:2018-12-12

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Abstract:

【Objective】 Soybean(Glycine max (L.) Merr.) seed generally form vitality from their physiological maturity period (R6 or R7 period). However, the seed is susceptible to high temperature and humidity (HTH) stress during this period. This will lead to a decline in seed vigor. The results will lay a foundation for further studying the mechanism of seed vigor formation under abiotic stress. 【Method】 Primer Premier 5.0 was used to design primers, and the full length cDNA sequence of GmLEA was isolated by using the cDNA of leaves of cv. Ningzhen No.1 and Xiangdou No.3 as template. The homologous amino acid sequence of GmLEA was searched by BLAST at NCBI, the protein sequences were multiple aligned using MEGA 6.0 and DNAMAN, and the phylogenetic tree was constructed using the N-J algorithm of MEGA 6.0. Yeast two-hybrid experiments were performed to verify the interaction of GmLEA and GmCDPKSK5 in yeast. A subcellular localization and bimolecular fluorescent complementation (BiFC) vector were constructed, The interaction between GmLEA and GmCDPKSK5 in tobacco leaf cells and the subcellular localization of the encoded protein were analyzed by gene-gun-mediated transformation of tobacco leaves. In addition, the tissue-specific expression of GmLEA gene and the expression pattern of GmLEA gene under HTH were analyzed by qRT-PCR, respectively. The pBI121 fusion expression vector was constructed and three homozygous overexpressed Arabidopsis lines were obtained through Agrobacterium- mediated method, and three independent homozygous T3 transgenic lines were used for analysis. 【Result】 The cDNA sequence of GmLEA gene contains a 1 377 bp open reading frame (ORF), and the subcellular localization result showed that the encoded protein was located on the cell membrane. The results of yeast two-hybrid rotation verification showed that GmLEA could interact with GmCDPKSK5 in yeast. In addition, bimolecular fluorescence complementation (BiFC) experiment showed that GmLEA could interact with GmCDPKSK5 on cell membrane of tobacco leaf cells. The results of tissue-specific analysis showed that GmLEA gene had higher expression levels in developing and mature seeds of both cultivars. The expression level of GmLEA was increased first and then decreased during the development of cv. Xiangdou No. 3 seeds. During the process of seed development of cv. Ningzhen No.1, the level of GmLEA expression was on the rise and reached the highest at 60 days after flowering. After high temperature and high humidity (HTH) stress, the expression of GmLEA was decreased at 96 h in cv. Xiangdou No. 3, and the other time points were increased. However, the expression was decreased at 24 h in cv. Ningzhen No.1. The germination potential, germination rate and seed vigor of GmLEA gene in transgenic Arabidopsis thaliana were significantly (P<0.01) higher than those of the wild type plants under HTH stress. 【Conclusion】 The GmLEA is involved in the formation of seed vigor under HTH stress,and has specific interaction with GmCDPKSK5, It is speculated that they may participate in the formation of seed vigor under HTH stress.

Key words: soybean, GmLEA, GmCDPKSK5, high temperature and high humidity stress, seed vigor

Table 1

Plant growth conditions"

类别
Class		 白天 Day 黑夜 Dark night 用途
Use
温度
Temperature
(℃)		 湿度
Humidity
(%)		 光照强度
Light intensity
(μmol·m-2·s-1)		 时间
Time
(h)		 温度
Temperature
(℃)		 湿度
Humidity
(%)		 光照强度
Light intensity
(μmol·m-2·s-1)		 时间
Time
(h)
对照
Control		 30 70 22000 10 25 60 0 14 GmLEA的表达分析Expression of GmLEA
处理
Treatment		 40 100 22000 10 30 70 0 14
对照
Control		 22 70 22000 16 20 60 0 8 种子生活力与活力指标的测定Determination of seed viability and vigor
处理
Treatment		 40 100 22000 16 35 80 0 8

Fig. 1

Phylogenetic tree of organism LEA protein from various organisms"

Fig. 2

Identification of the interaction between GmLEA and GmCDPKSK5 through yeast two hybrid"

Fig. 3

Subcellular localization of GmLEA protein in tobacco leaf cells"

Fig. 4

BiFC visualization of interaction between GmLEA and GmCDPKSK5 in tobacco leaf cells"

Fig. 5

The relative expression of GmLEA gene in soybean different organs Different uppercase letters indicate significant differences at P<0.01 levels"

Fig. 6

The relative expression of GmLEA gene in developing soybean seed"

Fig. 7

The expression patterns of the GmLEA gene in the seeds under HTH A: Xiangdou No.3; B: Ningzhen No.1. ** indicate that the differences are markly significant at 0.01 level"

Fig. 8

Effect on germination percentage of Arabidopsis seeds under HTH stress A: Illustration of representative 7-day-old seedlings from the developing seeds harvested from the wild type and transgenic line plants under the control and HTH treatment for 2 d; B: Germination percentage of the developing seeds harvested from the wild type and transgenic line plants under the control and HTH stress; C: Seed viability of the developing seeds harvested from the wild type and transgenic line plants under the control and HTH treatment, Bar=2 mm"

Table 2

The effect of HTH stress on germination of Arabidopsis seeds"

类别
Class		 株系
Lines		 发芽势
Germination potential (%)		 发芽指数
Germination index		 活力指数
Vitality index		 平均发芽天数
Average germination days (d)
处理
Treatment		 WT 10%±0.02D 10.88±0.30DE 5.52±0.25H 4.07±+0.02AB
121-GUS 9%±0.03D 7.51±0.39E 4.80±0.21I 4.02±0.03A
LEA-1 31%±0.07C 21.53±0.40C 9.88±0.14FG 3.92±0.04BC
LEA-2 32%±0.05C 22.62±0.41C 10.02±0.45F 3.91±0.06CD
LEA-3 34%±0.05C 20.08±0.27CD 9.93±0.23G 3.89±0.07BC
对照
Control		 WT 69%±0.08B 53.37±0.32AB 40.33±0.15E 3.59±0.02E
121-GUS 85%±0.01A 61.3±0.30A 41.74±0.22D 3.61±0.05E
LEA-1 72%±0.05B 51.74±0.45B 45.18±0.13B 3.69±0.04DE
LEA-2 73%±0.04AB 53.06±0.33AB 44.97±0.21C 3.67±+0.08E
LEA-3 75%±0.04AB 52.33±+0.29B 45.77±0.22A 3.70±0.03DE
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