Identification and Expression under Salt Stress of NHXs in Brassica napus L.
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摘要:
目的 系统鉴定甘蓝型油菜（Brassica napus L.）Na+/H+逆向转运蛋白（Na+/H+ antiporter, NHX）家族成员，筛选盐胁迫相关候选基因。 方法 利用公开的甘蓝型油菜品种中双11的基因组序列，通过同源比对的方法，在全基因组范围内获得NHX候选基因家族基因及蛋白序列，分析其理化性质、进化关系及盐胁迫下基因表达模式。 结果 共鉴定出21个甘蓝型油菜NHX基因家族成员，其氨基酸数量为71～ 1265 aa，等电点pI为5.54～7.68，内含子为0～24个；其中BnNHX1基因定位于细胞质膜，BnNHX5、BnNHX12定位于细胞膜及液泡膜，其余基因均只定位在液泡。NHX基因家族可分为3个亚家族，分布在11条染色体上，顺式作用元件分析表明NHX基因家族含有多种非生物胁迫响应元件。qRT-PCR结果发现BnNHXs基因受盐胁迫后，多数基因表达量上调，且叶片上的表达量整体高于根部，其中以BnNHX2、BnNHX6、BnNHX8、BnNHX11和BnNHX19等5个基因表达量变化相对较大。结论 BnNHX2、BnNHX6、BnNHX8、BnNHX11和BnNHX19等5个基因可作为油菜的耐盐候选功能基因，为下一步利用NHX基因开展油菜耐盐育种及耐盐分子机制研究奠定了基础。 Abstract:Objective Members of the Na+/H+ antiporter (NHX) family in Brassica napus L. were identified, and genes related to salt stress screened. Methods The genome of B. napus cv. Zhongshuang 11 was used to obtain the candidate genes, and NHXs sequences by homologous alignment. Physicochemical properties, evolutionary relationship, and expressions under salt stress of the genes were analyzed. Results Twenty-one BnNHXs were identified with 71 to 1265 amino acids, an isoelectric (pI) ranging from 5.54 to 7.68, and 0 to 24 introns. Among the BnNHXs, BnNHX1 was in the plasma membrane, BnNHX5 and BnNHX12 in the cell and vacuole membranes, and the remainders in the vacuole only. Phylogenetically, NHXs could be divided into 3 subfamilies scattered on 11 chromosomes containing a variety of abiotic stress response elements. The qRT-PCR expressions on most BnNHXs were upregulated under salt stress, with overall higher expression in the leaves than in the roots. Notably, BnNHX2, BnNHX6, BnNHX8, BnNHX11, and BnNHX19 had relatively more pronounced changes in the expression.Conclusion Five NHXs, namely, BnNHX2, BnNHX6, BnNHX8, BnNHX11, and BnNHX19, could be the candidate genes for breeding salt-tolerant new varieties as well as for studying molecular mechanisms of rapeseed. -
Key words:
- Brassica napus L. /
- NHX gene family /
- gene expression /
- salt stress
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图 1 甘蓝型油菜NHX基因家族系统发育及在基因组染色体分布
(a)油菜NHX基因家族系统进化树；（b）油菜NHX基因家族染色体定位。
Figure 1. Phylogeny tree and gene location in genome chromosome of BnNHXs
(a) phylogenetic tree of NHXs in rapeseed; (b) chromosomal mapping of NHXs in rapeseed.
图 2 甘蓝型油菜NHX基因家族成员蛋白motifs及基因结构分析
（a）油菜NHX基因家族预测保守蛋白基序；（b）油菜NHX基因家族基因结构；（c）预测的保守蛋白基序。
Figure 2. Protein motifs and gene structure of BnNHXs
(a) predicted conserved protein motif of rapeseed NHX gene family; (b) gene structure map of rapeseed NHX gene family; (c) predicted conserved protein motif sequence.
图 4 油菜NHXs基因盐胁迫条件下表达情况
(a) 油菜NHXs基因表达情况；(b) 部分基因的相对表达量。不同字母表示同一时间不同基因表达量之间显著差异（邓肯多重检验，P＜0.05）。
Figure 4. Expressions of BnNHXs under salt stress
(a)NHXs expressions in rapeseed; (b) relative expressions of some genes. Data with different letters indicate significant differences among different genes (Duncan multiple test, P＜0.05).
表 1 qRT-PCR分析引物序列
Table 1. Sequences of primers used for qRT-PCR
基因
Gene上游引物(5′-3′)
Forward primer (5′-3′)下游引物(5′-3′)
Reverse primer (5′-3′)BnNHX1 CGACCTCTTCGCATCCCACA GGGAGACAGCCACCAAGTGC BnNHX2 AGCAGCATTCGTCTTCCCAT TGCCCAGCACTTGTAAACTTG BnNHX3 TCAAAGGTCACTACCAAACACAC CAAGGAGTATCGCACTCACTCC BnNHX4 GGGCTTGGCACTGGAGTC AGATTATAGTCCCAATGGCGCC BnNHX5 CGGTTCGAGACCTGTAGATGCA AGTCCCGTACTCTAGAGATCCG BnNHX6 CTCTGGATTCAGTCTACAGCCC CTAGCACCCCAGTAACCATGGA BnNHX7 TGCTGCCGCCCATCATATTCC CAAGGTACACTAGCACGCCAGT BnNHX8 TTCGTTCATCCATCGGTTCTCCT CCAGAAGATGTGAGCTTTTGCC BnNHX9 GCGGCGACGCCGTATAGAC CGGAACTCTCGTCCCGCGA BnNHX10 CAGGCAATTGCACGTATTTGCG ACAGCTCCGACTAAAATGGCG BnNHX11 ACCGCCTTAATGATTGGGCT AATCGTCACGAAGTTGCGG BnNHX12 TACCGACCCTGTGGCTGT TCAGAGGTACTCCCCATCACC BnNHX13 GCGATGGCGACAGCAGTT GGCTCGGGAAGGAGTGCT BnNHX14 CGCCACGTCAGTTGTGCT CACCAAGCACGGTGCTCAA BnNHX15 TCGGGTTGATCGTTGGTG GACAATGGCTCCGAAGTTCG BnNHX16 CGCTTCTCATCGGGTTGATCGT GCTGAATCCTGACTGGGTGC BnNHX17 ACCAAAATCGATCCAGGTCC TCAGGGCGAGGCAAATTCTC BnNHX18 CAGTTGTTCACTTCAACGTCGT TTTTGAGGATTGTGCTTGTGGAG BnNHX19 GTCGGCCTCTGATCACGCC CAGCCCAATCAATAAGGCGG BnNHX20 CTGATGAATGATGGGACGGCA CAGCTCCAAGTGCGACTCTA BnNHX21 CCGCCATTGCAGTCGTAAGTT ACAGCTCCCTGTTTGCAACAT BnActin CTGGAATTGCTGACCGTATGAG ATCTGTTGGAAAGTGCTGAGGG 
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表 2 甘蓝型油菜NHX基因家族基本信息
Table 2. Molecular characteristics of NHXs in B. napus L.
基因名称
Gene name基因ID
Locus ID基因长度
Gene length/bp内含子数目
Introns number亚细胞定位
Subcellular localization蛋白理化性质 Putative Protein 氨基酸数量 aa 分子量 MW/Da 等电点 pI 亲水性 GRAVY BnNHX1 BnaA02T0312700ZS 835 3 细胞膜 200 22714.65 6.13 0.661 BnNHX2 BnaA02T0370900ZS 4476 13 液泡 544 60373.62 7.19 0.484 BnNHX3 BnaA05T0466800ZS 3877 13 液泡 558 61834.21 6.6 0.442 BnNHX4 BnaA05T0478300ZS 4057 14 液泡 548 60856.17 7.07 0.466 BnNHX5 BnaA06T0096600ZS 1123 4 细胞膜、液泡 195 20881.54 6.82 0.555 BnNHX6 BnaA07T0230000ZS 6884 24 液泡 1265 140509.11 6.5 0.138 BnNHX7 BnaA07T0383700ZS 4112 21 液泡 537 59711.75 5.64 0.359 BnNHX8 BnaA09T0055200ZS 4246 13 液泡 543 60165.4 7.67 0.499 BnNHX9 BnaA09T0219900ZS 2713 11 液泡 438 48477.01 7.68 0.615 BnNHX10 BnaA10T0114600ZS 2868 13 液泡 533 58931.9 6.87 0.537 BnNHX11 BnaC02T0497000ZS 4378 13 液泡 544 60387.65 7.19 0.488 BnNHX12 BnaC05T0118600ZS 5234 18 细胞膜、液泡 743 82543.98 6.95 0.303 BnNHX13 BnaC05T0526800ZS 3819 13 液泡 560 62245.77 6.6 0.43 BnNHX14 BnaC05T0540200ZS 4076 14 液泡 549 60991.34 7.09 0.475 BnNHX15 BnaC06T0248200ZS 3960 20 液泡 557 62369.9 5.54 0.373 BnNHX16 BnaC06T0451400ZS 3949 19 液泡 552 61687.26 5.93 0.365 BnNHX17 BnaC07T0342400ZS 2235 9 液泡 471 52583.11 6.82 0.376 BnNHX18 BnaC07T0518600ZS 216 0 液泡 71 7960.31 5.85 0.683 BnNHX19 BnaC09T0042200ZS 4345 12 液泡 535 59317.32 7.19 0.491 BnNHX20 BnaC09T0255800ZS 2605 11 液泡 385 41965.58 7.15 0.738 BnNHX21 BnaC09T0380600ZS 2813 12 液泡 541 60042.5 7.57 0.598
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