Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (1): 18-28.doi: 10.3864/j.issn.0578-1752.2020.01.002

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

The Mechanism of Ectopic Expression of Brassica juncea Multidrug and Toxic Compound Extrusion (BjMATE) to Enhance the Resistance to Acid and Aluminum Stress in Alfalfa

XiaoDong LI1,2,YiShun SHANG1(),ShiGe LI1,2,GuangJi CHEN1,2,ChengJiang PEI1,2,Fang SUN1,XianQin XIONG1   

  1. 1 Guizhou Institute of Prataculture, Guizhou Academy of Agricultural Science, Guiyang 550006
    2 Guizhou Dingxin Agriculture and Animal Husbandry Technology Co. Ltd, Guiyang 550006
  • Received:2019-06-03 Accepted:2019-09-10 Online:2020-01-01 Published:2020-01-19
  • Contact: YiShun SHANG E-mail:2892486467@qq.com

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Abstract:

【Objective】 Acid and aluminum stress is one of the major environmental stresses in agricultural production in the south of China. Studying the mechanism of native plants adaptation to acid and aluminum stress, and identifying and making use of the excellent gene resources would benefit to breed new varieties of acid and aluminum resistant crops, that will play a fundamental role in molecular breeding of alfalfa. 【Method】 In previous study, full length of BjMATE was cloned from Pingba bitter rape, a native Brassica juncea resource in Guizhou province, and the overexpression vector was constructed by the alfalfa genetic improvement group of Guizhou Institute of Prataculture. The transgenic plants of alfalfa and Arabidopsis thaliana were obtained by tissue culture and flower dipping methods, respectively. Molecular examination was carried out with NPT resistance gene primers and gene-specific primers; and the expression of BjMATE in transgenic plants was examined by qRT-PCR. The germination rate and seedling growth of transgenic and control alfalfa lines were analyzed by hydroponic method under acid, aluminum and acid aluminum combination stress conditions. The alteration of the seedling and root growth was also analyzed between transgenic and Zhongmu 1# alfalfa under a long term but weaker acid-aluminum stress condition. Antioxidant enzyme activity, including POD, SOD, CAT, and MDA concentration alterations between OEMs-5 and Medicago sativa Zhongmu 1# were analyzed with microplate reader under acid, aluminum and acid-aluminum combined stress conditions. At the same time, expression changes of AtMATE, AtPIN2, AtALS3, AtALMT1, and AtSTOP1 which were key genes involving in acid aluminum regulation pathways, were analyzed in the model plant Arabidopsis thaliana by qRT-PCR, and potential regulation network were discussed. 【Result】 The positive rates of transgenic Arabidopsis and alfalfa were 100% and 66.7%, respectively. Compared with the control lines, the expression levels of the candidate transgenic alfalfa and Arabidopsis lines were up-regulated by 63.02 and 76.87 times, respectively. There was no significant difference in seed germination rate between OEMs-5 and Zhongmu 1# under normal, acid stress, aluminum ion stress and acid-aluminum combination stress conditions. While the germination potential was significantly different among the treatments, as well as the materials, OEMs-5 was significantly better than Zhongmu 1#. After a long-term weak stress treatment, the plant height of OEMs-5 was not significantly different from that of Zhongmu 1#; but the biomass and root length of OEMs-5 was significantly better than that of Zhongmu 1# under aluminum stress and acid-aluminum combined stress conditions. Antioxidant enzyme activity and MDA concentration alterations between OEMs-5 and Zhongmu 1# were different. POD and SOD activity increased in both OEMs-5 and Zhongmu 1# after stress treatment, however, significant difference was only detected in SOD assay. No significant difference in CAT activity was detected between the two materials, and the treatments. The content of MDA decreased slightly, significant differences were detected between OEMs-5 and Zhongmu 1# under aluminum stress and acid-aluminum combined stress conditions. Expressions of AtMATE, AtPIN2, AtALS3, AtALMT1 and AtSTOP1 were up-regulated under aluminum stress and acid-aluminum combined stress in both OEMs-5 and Zhongmu 1#, however, no significant difference were detected between them except AtSTOP1.【Conclusion】BjMATE can positively regulate alfalfa tolerance to acid-aluminum stress during seed germination and seedling growth stages. Reactive oxygen species (ROS) scavenger system, especially the activity of SOD, and AtSTOP1 gene which is a key gene involving in acid-aluminum stress regulation pathway, may participate in BjMATE mediated acid-aluminum stress regulation.

Key words: acid aluminum stress, MATE, alfalfa, gene expression regulation

Table 1

Primer used in the real time PCR assay"

基因位点Gene locus 序列Sequence(5′-3′) 用途Purpose
NPT F：GTGCCCTGAATGAACTGC 抗性基因PCR检测
R：CAATATCACGGGTAGCCA PCR with resistance gene primer
35S F：TCCCACTATCCTTCGCAAG 基因特异性引物检测
MATE R：TCAGACCGACGCATTTATCTTT Gene specific primer examination
qBjMATE F：AAGGCGTTCTATCAGGAGTG BjMATE表达分析
R：GAGTGATGAAGATTGGGAAA qRT-PCR for BjMATE
qAtMATE F：CTCTTGAGTTTCATGGGAGTA AtMATE表达分析
R：CGGGTCGAGTATTATGTTTG qRT-PCR for AtMATE
qAtPIN2 F：CGAGTGGAGCAAGTGGAGTC AtPIN2表达分析
R：GTGGATACATCGGTGGAAGA qRT-PCR for AtPIN2
qAtALS3 F：AATGTTCTTGCTCGTCCTCC AtALS3表达分析
R：CTTGTCTTGGCGTTGCTCCT qRT-PCR for AtALS3
qAtALMT1 F：TCATTATTTGAGGAGCAGTC AtALMT1表达分析
R：AAAGAGTAACGCAAAGGAAA qRT-PCR for AtALMT1
qAtSTOP1 F：TTCATCAGACTGTGGGAATT AtSTOP1表达分析
R：AGGTTACTCAACATCGTCCT qRT-PCR for AtSTOP1
qMsGPDH F：ACAAACATGGGAGCATCCTTACTAG qRT-PCR（紫花苜蓿内参）
R：GTTTTTACCGACAAGGACAAAGCT Reference gene in alfalfa
qAtActin7 F：GATATTCAGCCACTTGTCTGTGAC qRT-PCR（拟南芥内参）
R：CATGTTCGATTGGATACTTCAGAG Reference gene in Arabidopsis

Fig. 1

Molecular detection and gene expression analysis of transgenic alfalfa and Arabidopsis A: Transgenic alfalfa examination with NPT and BjMATE primers; B: Transgenic Arabidopsis examination with NPT and BjMATE primes. C: qRT-PCR analysis of BjMATE in transgenic alfalfa; D: qRT-PCR analysis of BjMATE in transgenic Arabidopsis. * indicate a significant difference were detected at P<0.05 level"

Fig. 2

Seed germination and root length of OEMs-5 and Zhongmu 1# under acid-aluminum stress conditions A: Seedlings of OEMs-5 and Zhongmu 1# germinated from seeds after acid-aluminum stress treatment, Bar=1 cm; B: Germination rate of OEMs-5 and Zhongmu 1#; C: Root length of OEMs-5 and Zhongmu 1#; e indicate a significant difference was detected at P<0.05 level between OEMs-5 and Zhongmu 1# lines, while E indicate a significant difference was detected at P<0.05 level between stress treatment and control within the same plant line. The same as below"

Fig. 3

Plant height and root length of OEMs-5 and Zhongmu 1# after a long time treatment under a weak acid-aluminum stress condition A: Seedlings of OEMs-5 and Zhongmu 1# after a long time treatment under a weak acid-aluminum stress condition, Bar=1 cm; B: Plant height of OEMs-5 and Zhongmu 1#; C: Biomass of OEMs-5 and Zhongmu 1#; D: Root length of OEMs-5 and Zhongmu 1#"

Fig. 4

Examination of physiological parameter in OEMs-5 and Zhongmu 1# after acid-aluminum stress treatment A-D: Alterations of POD (A), SOD (B), CAT (C) and MDA (D) between OEMs-5 and Zhongmu 1# after acid-aluminum stress treatment"

Fig. 5

Acid-aluminum stress related genes expression analysis in OEAt-1 A: Expression analysis of BjMATE in OEAt-1 under normal (N), acid (H+), aluminum ion (Al3+) and acid-aluminum combined stress (H+ & Al3+) conditions; B: Acid-aluminum regulation maker genes expression alterations in OEAt-1 and wild type Arabidopsis seedlings after stress treatments"

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