草业学报 ›› 2022, Vol. 31 ›› Issue (5): 124-134.DOI: 10.11686/cyxb2021087
• 研究论文 • 上一篇
刘亚男1(), 于人杰2, 高燕丽3, 康俊梅1, 杨青川1, 武志海2(), 王珍1()
收稿日期:
2021-03-15
修回日期:
2021-03-25
出版日期:
2022-05-20
发布日期:
2022-03-30
通讯作者:
武志海,王珍
作者简介:
Corresponding author. E-mail: wangzhen@caas.cn, wuzhihai1116@163.com基金资助:
Ya-nan LIU1(), Ren-jie YU2, Yan-li GAO3, Jun-mei KANG1, Qing-chuan YANG1, Zhi-hai WU2(), Zhen WANG1()
Received:
2021-03-15
Revised:
2021-03-25
Online:
2022-05-20
Published:
2022-03-30
Contact:
Zhi-hai WU,Zhen WANG
摘要:
膜联蛋白(annexins)是一类进化保守的多基因家族蛋白,它们广泛存在于真核生物中,能通过Ca2+与膜磷脂的结合参与胁迫相关的多种生物学过程。早期对膜联蛋白的研究多集中于脊椎动物,对植物膜联蛋白的认识开始于番茄。关于豆科植物尤其是牧草中膜联蛋白的研究还鲜有报道。本研究分析了蒺藜苜蓿膜联蛋白与饲草紫花苜蓿同源蛋白的进化关系,研究了蒺藜苜蓿膜联蛋白基因MtANN2的表达模式,进一步利用拟南芥同源基因的突变体阐明了MtANN2在根系发育和盐胁迫中的功能。RT-qPCR结果显示,MtANN2在根中高丰度表达,且表达水平受NaCl诱导。RNA原位杂交表明MtANN2特异表达于幼苗侧根原基。拟南芥同源基因AtANN2的T-DNA插入突变体植株弱小、侧根数少、根鲜重低,且对盐(100 mmol·L-1)处理的敏感性显著高于野生型。超表达MtANN2于atann2后转基因植株的侧根数介于野生型与突变体之间,根鲜重接近野生型,表明MtANN2能在一定程度上互补该突变体的表型缺陷。在盐处理下,该转基因株系的发芽率和长势均恢复到类似野生型的水平。以上结果从分子水平上表明,蒺藜苜蓿膜联蛋白MtANN2参与植物根系生长及盐胁迫响应,高水平表达该基因能够改善植物的耐盐性。本研究为紫花苜蓿耐盐分子育种提供了备选基因。
刘亚男, 于人杰, 高燕丽, 康俊梅, 杨青川, 武志海, 王珍. 蒺藜苜蓿膜联蛋白MtANN2基因的表达模式及盐胁迫下的功能分析[J]. 草业学报, 2022, 31(5): 124-134.
Ya-nan LIU, Ren-jie YU, Yan-li GAO, Jun-mei KANG, Qing-chuan YANG, Zhi-hai WU, Zhen WANG. Expression pattern and biological functions of an annexin encoding gene MtANN2 in Medicago truncatula under salt stress[J]. Acta Prataculturae Sinica, 2022, 31(5): 124-134.
引物名称Primer | 序列5′-3′ Sequence 5′-3′ |
---|---|
MtANN2 F | ATGGCGACATTGAAGATC |
MtANN2 R | CTACTCATCACGTCCCATCAG |
1302-MtANN2 F | gaacacgggggactcttgaccATGGCGACATTGAAGATC |
1302-MtANN2 R | ctcctttactagtcagatctacCATCTCATCACGTCCCATCAG |
35S F | CTATCCTTCGCAAGACCCTTC |
1302 R | TTCCGTATGTTGCATCACCTT |
LP | GATTGTGAAACTGTTGTATTTGGTG |
RP | GATTGCGGAAGCTGTAGTTGC |
T-DNA RB | GTGGATTGATGTGATATCTCC |
qRT-Atactin F | TCCATCGATTGTTCACAGGA |
qRT-Atactin R | TCACCACCACGAACCAGATA |
qRT-MtANN2 F | GCGGTGCTTTTCAAGGATGG |
qRT-MtANN2 R | ACCTTACGCTGAGCTGCATT |
qRT-AtANN2 F | TCTTGTGAGCACTTTCAGGTAT |
qRT-AtANN2 R | AGTTCTTGTTAATGGCGTTTCC |
MtANN2-in situ F | TAATACGACTCACTATAGGGTAGCTAATCAAGCAACTAAAATG |
MtANN2-in situ R | ATTTAGGTGACACTATAGAATAGGGACTTTTTGAAACGGGCTTG |
表1 试验中所用引物
Table 1 Primers used in the study
引物名称Primer | 序列5′-3′ Sequence 5′-3′ |
---|---|
MtANN2 F | ATGGCGACATTGAAGATC |
MtANN2 R | CTACTCATCACGTCCCATCAG |
1302-MtANN2 F | gaacacgggggactcttgaccATGGCGACATTGAAGATC |
1302-MtANN2 R | ctcctttactagtcagatctacCATCTCATCACGTCCCATCAG |
35S F | CTATCCTTCGCAAGACCCTTC |
1302 R | TTCCGTATGTTGCATCACCTT |
LP | GATTGTGAAACTGTTGTATTTGGTG |
RP | GATTGCGGAAGCTGTAGTTGC |
T-DNA RB | GTGGATTGATGTGATATCTCC |
qRT-Atactin F | TCCATCGATTGTTCACAGGA |
qRT-Atactin R | TCACCACCACGAACCAGATA |
qRT-MtANN2 F | GCGGTGCTTTTCAAGGATGG |
qRT-MtANN2 R | ACCTTACGCTGAGCTGCATT |
qRT-AtANN2 F | TCTTGTGAGCACTTTCAGGTAT |
qRT-AtANN2 R | AGTTCTTGTTAATGGCGTTTCC |
MtANN2-in situ F | TAATACGACTCACTATAGGGTAGCTAATCAAGCAACTAAAATG |
MtANN2-in situ R | ATTTAGGTGACACTATAGAATAGGGACTTTTTGAAACGGGCTTG |
图1 拟南芥和2种苜蓿中膜联蛋白的进化关系分析红色圆点代表蒺藜苜蓿膜联蛋白,蓝色代表拟南芥膜联蛋白,黑色代表紫花苜蓿膜联蛋白。Red dots represent annexins in M. truncatula, blue for annexins in A. thaliana, and black for annexins in M. sativa.
Fig.1 Phylogenetic analysis of annexin proteins from Arabidopsis and two Medicago species
图2 MtANN2的表达模式分析A:RT-qPCR分析MtANN2在蒺藜苜蓿根及幼苗中的表达量;B:原位杂交分析MtANN2在根系组织中的表达。其中,a、b为反义探针杂交结果,c、d为正义探针杂交结果;a、c为根系横切,b、d为根系纵切;箭头表示侧根原基。其中,正义探针作为阴性对照;C:MtANN2对NaCl(200 mmol·L-1)处理的响应分析。标准误差来自3个生物重复,数据利用单因素方差分析及邓肯检验,不同小写字母代表差异显著(P<0.05)。下同。A: The relative expression of MtANN2 in roots and aerial tissues of M. truncatula; B: In situ hybridization of MtANN2 in roots of M. truncatula. a, b: Hybridization by the anti-sense probe; c, d:Hybridization by the sense probe; a, c: Root transection; b, d: Root longitudinal section; Arrows indicate lateral root primordial; Sense probe acts as a negative control; C: The relative expression of MtANN2 under NaCl treatment (200 mmol·L-1). Error bar represents mean±SD of three biological replicates. Statistical analysis was performed by ANOVA with Duncan’s test. Lowercase letters represent significant difference (P<0.05). The same below.
Fig.2 Analysis of the expression pattern of MtANN2 in M. truncatula
图4 atann2突变体的鉴定及表型分析A:3周龄拟南芥;B:拟南芥AtANN2基因结构及atann2 T-DNA插入位点示意图,黑色块表示外显子,灰色块为非编码区(UTR),黑线为内含子,箭头代表基因型鉴定引物的位置,RB代表T-DNA的右边界,LP代表基因组侧翼序列上的上游引物,RP代表基因组侧翼序列上的下游引物,ATG代表起始密码子;C:atann2突变体基因型鉴定,Marker为分子大小标记;D:AtANN2表达水平分析;E:正常条件和盐处理(100 mmol·L-1 NaCl)下根系生长比较;F:相对一级侧根数统计;G:相对根长统计;H:相对根鲜重统计。图中列出盐处理后各指标减少的百分数,WT代表野生型,下同。A: Representative image of 3-week-old Arabidopsis seedlings; B: Schematic diagram of AtANN2 and the T-DNA insertion line of atann2, the boxes in black and gray represent exons and untranslated region, respectively, solid line indicates intron and arrow represents the location of primers used in screening; RB represents the right border of T-DNA, LP represents the forward primer of the genomic franking sequence, RP represents the reverse primer of the genomic franking sequence, ATG represents the start codon; C: PCR screening of the homozygous atann2; Marker represents the reference for DNA molecular size;D: Relative expression of AtANN2;E: Response to NaCl treatment;F: Statistical analysis of the relative No. of lateral roots; G: The relative length of roots; H: The relative fresh weight of roots. The subtraction between treatment and control was indicated by percentage, WT indicates wild types, the same below.
Fig.4 Screening and phenotypic analysis of atann2 mutant
图5 异源表达MtANN2于拟南芥atann2转基因材料转录水平鉴定由于是异源表达,因此以阳性转基因株系中表达量最低者为1,从而计算其余株系的相对表达水平。The expression of the positive line with the lowest expression level was regarded as 1 to calculate the relative expression of others.
Fig.5 Identification of transcriptional level of MtANN2 in transgenic atann2
图6 异源表达MtANN2的atann2拟南芥对NaCl的响应A:盐处理(100 mmol·L-1 NaCl)下的发芽率比较,*代表该时间点atann2发芽率与其他株系差异显著(P<0.05);B:盐处理(100 mmol·L-1 NaCl)下的根系生长比较。A: Comparison of germination rate of the four genotypes under salt treatment (100 mmol·L-1 NaCl), * represents the significantly different germination rate of atann2 (P<0.05); B: Comparison of root growth of the four genotypeswith and without NaCl treatment.
Fig.6 Analysis of atann2 ectopically expressing MtANN2 under NaCl treatment
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