向日葵HaACO1基因的表达分析及功能验证

doi:10.13560/j.cnki.biotech.bull.1985.2020-1482

摘要/Abstract

摘要：

为加强对向日葵ACC氧化酶基因的利用,以前期从盐诱导的向日葵中克隆的ACC氧化酶基因HaACO1（GenBank accession number. KP966508）为对象,进行了该基因在不同胁迫条件下的表达分析及在烟草中的超表达研究。结果表明,该基因受病原菌、机械损伤、低温、NaCl和水杨酸胁迫诱导表达,且在不同的胁迫下表现出不同的表达模式;HaACO1在向日葵根、下胚轴和叶中均有表达,但在叶中的表达量最高,在根中的表达量最低。利用瞬时表达载体进行亚细胞定位分析,发现HaACO1-GFP在洋葱表皮细胞的细胞质中有表达。构建HaACO1植物表达载体进行过表达分析,表明在含有NaCl的分化培养基上,转基因烟草叶色失绿程度较野生型的轻,分化能力较野生型的高;低温、干旱和NaCl胁迫下,转基因烟草的HaACO1相对表达量高于野生型;NaCl胁迫下,转基因烟草的可溶性蛋白（soluble protein）、脯氨酸（proline,PRO）和叶绿素（chlorophyll）含量及过氧化物酶（peroxidase,POD）和超氧化物歧化酶（superoxide dismutase,SOD）活性提高;脯氨酸合成关键酶基因P5CS及抗氧化相关基因POD、MnSOD和GuZnSOD表达上调。HaACO1过表达提高了烟草的耐盐性,这将为进一步理解向日葵耐盐分子机制以及利用该基因进行作物抗逆性状改良奠定基础。

关键词: 向日葵, HaACO1, 胁迫应答, 亚细胞定位, 过表达

Abstract:

To exploit genes encoding ACC oxidase in sunflower more profitably,we studied one ACC oxidase gene HaACO1（GenBank accession number：KP966508）cloned from previous salt-induced sunflower plants. The expression patterns of HaACO1 under different stress types were investigated. Additionally,the overexpression of this gene in tobacco was carried out. The results showed that the expression of HaACO1 in sunflower was induced by pathogens,mechanical damage,low temperature,NaCl and salicylic acid stress,and expression patterns were distinct under these types of stress. HaACO1 expressed in the roots,hypocotyls and leaves of sunflower plants,with the highest expression in the leaves and the lowest expression in the roots. By taking advantage of a transient expressive vector,the subcellular localization of HaACO1-GFP was found to be largely in the cytoplasm of onion epidermal cells. A plant vector of expressing HaACO1 was constructed to analyze the overexpressing of HaACO1. The results indicated the decrease in green color of leaves of transgenic tobacco was smaller than that of the wild plants placed on differentiation medium with NaCl,while the leaf differentiation was higher than that of the wild plants. Under low temperature,drought and NaCl stresses,the relative expression of HaACO1 in the transgenic tobacco plants was higher than that of the wild plants. During NaCl stress,the contents of total soluble protein,proline and chlorophyll and the activities of peroxidase and superoxide dismutase in the transgenic tobacco plants increased;and the expression of a key gene P5CS for proline biosynthesis and three antioxidant related genes POD,MnSOD and CuZnSOD were all up-regulated. These results indicate that overexpression of HaACO1 in tobacco plants enhances the plants’ tolerance to high concentrations of NaCl,providing foundations for further understanding the molecular mechanism of salt tolerance in sunflower,and laying a foundation for using this gene in crop stress resistance improvement.

Key words: sunflower, HaACO1, stress response, subcellular localization, overexpression

孙瑞芬, 张艳芳, 牛素清, 郭树春, 李素萍, 于海峰, 聂惠, 牟英男. 向日葵HaACO1基因的表达分析及功能验证[J]. 生物技术通报, 2021, 37(9): 114-124.

SUN Rui-fen, ZHANG Yan-fang, NIU Su-qing, GUO Shu-chun, LI Su-ping, YU Hai-feng, NIE Hui, MOU Ying-nan. Expression Analysis and Functional Verification of the HaACO1 Gene in Sunflower[J]. Biotechnology Bulletin, 2021, 37(9): 114-124.

图/表 10

表1 引物信息

Table 1 Primer information

名称 Name	编号 No.	序列 Sequence（5'-3'）	用途 Application
qHaACO1	F1	GCTTCAAAGAAATGGTGGCT	HaACO1的qRT-PCR qRT-PCR of HaACO1
qHaACO1	R1	GGGAGATGGCGGAGATAGA	HaACO1的qRT-PCR qRT-PCR of HaACO1
18S rRNA	F2	AGAAACGGCTACCACATCCA	向日葵内参基因18S rRNA Reference gene 18S rRNA in sunflower
18S rRNA	R2	TTGTTATTTATTGTCACTACCTCCC	向日葵内参基因18S rRNA Reference gene 18S rRNA in sunflower
sHaACO1	F3	CGGGGTACCATGGAGGAGGAGACATTTCCAG Kpn I	HaACO1编码区扩增（引入KpnⅠ/BamⅠ位点） Amplification of HaACO1 coding region（carrying KpnⅠ/BamH I sites）
sHaACO1	R3	CGGGATCCAGCAGTTGCAATGGGGTC Bam Ⅰ
zHaACO1	F4	CGGGATCCATGGAGGAGGAGACATTTCCAG Bam HI	HaACO1编码区扩增（引入Bam HⅠ/KpnⅠ位点） Amplification of HaACO1 coding region（carrying BamHⅠ/KpnⅠ sites）
zHaACO1	R4	CGGGGTACCTTAAGCAGTTGCAATGGGGTC Kpn I
CaMV35S	p-F	AGACGTTCCAACCACGTCTTCA	转基因烟草中HaACO1基因检测（CaMV 35S启动子近3'端序列） HaACO1 detection in transgenic tobacco（near 3' terminal sequence of CaMV35S promoter）
NtEF-1α	F5	TGAGATGCACCACGAAGCTC	烟草内参基因EF-1α Reference gene EF-1α in tobacco
NtEF-1α	R5	CCAACATTGTCACCAGGAAGTG	烟草内参基因EF-1α Reference gene EF-1α in tobacco
P5CS	F6	TGGTCGTCAGCGGCTTAGAT	烟草P5CS的qRT-PCR qRT-PCR of P5CS in tobacco
P5CS	R6	TGCCAAACTGTCATTGTCCC	烟草P5CS的qRT-PCR qRT-PCR of P5CS in tobacco
POD	F7	CCCTGGTGTTGTTTCTTGTG	烟草POD的qRT-PCR qRT-PCR of POD in tobacco
POD	R7	CCTGAGCCTGAACTTCTTGG	烟草POD的qRT-PCR qRT-PCR of POD in tobacco
MnSOD	F8	GCAGACGGACCTTAGCAACA	烟草MnSOD的qRT-PCR qRT-PCR of MnSOD in tobacco
MnSOD	R8	GGGAGCCAAAGTTAGTGTCG	烟草MnSOD的qRT-PCR qRT-PCR of MnSOD in tobacco
GuZnSOD	F9	CGGGACCACATTACAATCCT	烟草GuZnSOD的qRT-PCR qRT-PCR of GuZnSOD in tobacco
GuZnSOD	R9	ATCAGCGTGAACAACCACAG	烟草GuZnSOD的qRT-PCR qRT-PCR of GuZnSOD in tobacco

图1 HaACO1在向日葵不同器官的表达分析 **：相对于根差异极显著（P<0.01）

Fig.1 Expression of HaACO1 in different organs of sunflower **：Extremely significant difference compared with root（P<0.01）

图2 HaACO1在黄萎病病原菌V21诱导下的表达分析 **：相对于0 d差异极显著（P<0.01）

Fig.2 HaACO1 expression under the induction of Verticillium wilt pathogen V21 **：Extremely significant difference compared with 0 d（P<0.01）

图3 非生物胁迫的不同时间点HaACO1相对表达分析 N：NaCl胁迫;J：机械损伤胁迫;T：低温胁迫;S：水杨酸胁迫;*,**：分别为相对于0 d差异显著（P<0.05）和极显著（P<0.01）

Fig.3 HaACO1 expression at different time under different abiotic stresses N：NaCl stress. J：Mechanical damage stress. T：Low temperature stress. S：Salicylic acid stress. *,**：Significant difference（P<0.05）and extremely significant difference（P<0.01）,respectively,compared with 0 d

图4 融合蛋白HaACO1-GFP在洋葱表皮细胞中定位

Fig. 4 Localization of HaACO1-GFP fusions in onion epidermal cell

图5 抗性植株的分子检测 A：PCR;B：RT-PCR. CK+：重组质粒pPZP221-HaACO1;CK-：WT;1-11：转化植株

Fig.5 Molecular test of resistant plants with PCR and RT-PCR CK+：Recombinant plasmid pPZP221-HaACO1. CK-：Wild type（WT）. 1-11：Transformed plants

图6 盐胁迫对烟草叶片离体培养的影响

Fig.6 Effect of salt stress on in vitro culture of tobacco leaves

图7 非生物胁迫条件下转基因烟草中HaACO1的表达分析 CK：未处理;DW：4℃低温处理;PEG：20% PEG6000处理;NaCl：120 mmol/L处理;**：胁迫条件下转基因株系T-10相对于WT差异极显著（P<0.01）

Fig. 7 HaACO1 expression analysis in transgenic plants under abiotic stresses CK: Untreatment; DW: lower temperature treatment under 4℃; PEG: the treatment with 20% PEG6000; NaCl: the treatment with 120 mol/L NaCl; **: extremely significant difference of transgenetic line T-10 compared with WT under stress （P<0.01）

图8 转基因烟草中抗逆相关生理生化指标分析 **：NaCl胁迫下转基因株系T-10相对于WT差异极显著（P<0.01）,下同

Fig.8 Analyses of physiological parameters related to stress tolerance in transgenic tobacco **：Extremely significant difference of transgenic line T-11 compared with WT under stress（P<0.01）. The same below

图9 转基因烟草中胁迫相关基因的表达分析

Fig. 9 Expression analysis of stress-responsive genes in transgenic tobacco

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