Expression Analysis and Functional Verification of the HaACO1 Gene in Sunflower

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

Abstract

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

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.

Figures/Tables 10

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

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

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

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

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

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

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）

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

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

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