Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (6): 190-202.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0012
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LI Bo-jing(), ZHENG La-mei, WU Wu-yun, GAO Fei(), ZHOU Yi-jun()
Received:
2024-01-04
Online:
2024-06-26
Published:
2024-06-24
Contact:
GAO Fei, ZHOU Yi-jun
E-mail:c2236363710@163.com;gaofei@muc.edu.cn;zhouyijun@muc.edu.cn
LI Bo-jing, ZHENG La-mei, WU Wu-yun, GAO Fei, ZHOU Yi-jun. Evolution, Expression, and Functional Analysis of the HSP20 Gene Family from Simmondisa chinensis[J]. Biotechnology Bulletin, 2024, 40(6): 190-202.
基因名称Gene name | 上游引物 Forward primer(5'-3') | 下游引物 Reverse primer(5'-3') |
---|---|---|
18S | cgttaacgaacgagacctca | cccagaacatctaagggcat |
ScHSP20-1 | gttgcggtcgcatttgttg | cgtccacggaagccatagaa |
ScHSP20-3 | gccctgaaaatgaagagggtac | cctttgaacttggcttggatg |
ScHSP20-4 | aatcaaaggagaaggggagaaa | aggaaccaccaccttcaacact |
ScHSP20-13 | cgtcaagagggactcaccaaa | caccagaaaccgaaggagga |
ScHSP20-14 | tcaagagggactcaccaaacag | agccacgtaagccccaatc |
ScHSP20-16 | acacccttcatcacatcctcg | agctgttggggtactccttcac |
ScHSP20-17 | accgtgtagagcggagcagt | cagtgacagtcaggaccccat |
ScHSP20-20 | cagtaagagggcggaggatg | aagggaaagacgacgaggc |
ScHSP20-25 | atggcgagcaaggcgttgacatgca | tgcatgtcaacgccttgctcgccat |
ScHSP20-28 | tggcctctgtacgtgctgatt | caccctgttctcctccacttct |
ScHSP20-23 | tgaggaacctccgacaaagac | cctcctgatgttgacgatgaaa |
ScHSP20-26 | tccaggtcttagcaaggagca | tgtctttccctgtgggttcg |
ScHSP20-32 | ctccctctgactgggtttcg | gggcgttctccacttatcctta |
Table 1 Primers used in RT-qPCR
基因名称Gene name | 上游引物 Forward primer(5'-3') | 下游引物 Reverse primer(5'-3') |
---|---|---|
18S | cgttaacgaacgagacctca | cccagaacatctaagggcat |
ScHSP20-1 | gttgcggtcgcatttgttg | cgtccacggaagccatagaa |
ScHSP20-3 | gccctgaaaatgaagagggtac | cctttgaacttggcttggatg |
ScHSP20-4 | aatcaaaggagaaggggagaaa | aggaaccaccaccttcaacact |
ScHSP20-13 | cgtcaagagggactcaccaaa | caccagaaaccgaaggagga |
ScHSP20-14 | tcaagagggactcaccaaacag | agccacgtaagccccaatc |
ScHSP20-16 | acacccttcatcacatcctcg | agctgttggggtactccttcac |
ScHSP20-17 | accgtgtagagcggagcagt | cagtgacagtcaggaccccat |
ScHSP20-20 | cagtaagagggcggaggatg | aagggaaagacgacgaggc |
ScHSP20-25 | atggcgagcaaggcgttgacatgca | tgcatgtcaacgccttgctcgccat |
ScHSP20-28 | tggcctctgtacgtgctgatt | caccctgttctcctccacttct |
ScHSP20-23 | tgaggaacctccgacaaagac | cctcctgatgttgacgatgaaa |
ScHSP20-26 | tccaggtcttagcaaggagca | tgtctttccctgtgggttcg |
ScHSP20-32 | ctccctctgactgggtttcg | gggcgttctccacttatcctta |
用途 Purpose | 基因名称 Gene name | 上游引物 Forward primer(5'-3') | 下游引物 Reverse primer(5'-3') |
---|---|---|---|
原核表达载体 Prokaryotic expression vector | ScHSP20-17 | cgcgtggatccccggATGTCGCTTATTCCAAGTAGGT | agtcagtcacgatgcACCAGAGATTTCAATGGCTTTG |
酵母表达载体 Yeast expression vector | ScHSP20-17 | ctatagggaatattaATGTCGCTTATTCCAAGTAGGT | cggccgttactagtgACCAGAGATTTCAATGGCTTTG |
亚细胞定位载体 Subcellular localization vector | ScHSP20-17 | agtccggagctagctATGTCGCTTATTCCAAGTAGGT | cccttgctcaccatgACCAGAGATTTCAATGGCTTTG |
内参基因 Internal reference gene | Actin3 | GAGGGCCGTGTTCCCCAGCATCGTC | TCTTTTTGATTGAGCCTCATCCCCT |
基因表达分析 Gene expression analysis | ScHSP20-17 | AAGAATGATACCTGGCACCG | TGACCTCCTCCTTAGGCACA |
Table 2 Primer sequences
用途 Purpose | 基因名称 Gene name | 上游引物 Forward primer(5'-3') | 下游引物 Reverse primer(5'-3') |
---|---|---|---|
原核表达载体 Prokaryotic expression vector | ScHSP20-17 | cgcgtggatccccggATGTCGCTTATTCCAAGTAGGT | agtcagtcacgatgcACCAGAGATTTCAATGGCTTTG |
酵母表达载体 Yeast expression vector | ScHSP20-17 | ctatagggaatattaATGTCGCTTATTCCAAGTAGGT | cggccgttactagtgACCAGAGATTTCAATGGCTTTG |
亚细胞定位载体 Subcellular localization vector | ScHSP20-17 | agtccggagctagctATGTCGCTTATTCCAAGTAGGT | cccttgctcaccatgACCAGAGATTTCAATGGCTTTG |
内参基因 Internal reference gene | Actin3 | GAGGGCCGTGTTCCCCAGCATCGTC | TCTTTTTGATTGAGCCTCATCCCCT |
基因表达分析 Gene expression analysis | ScHSP20-17 | AAGAATGATACCTGGCACCG | TGACCTCCTCCTTAGGCACA |
Fig. 3 Evolutionary analysis of the HSP20 gene in Arabidopsis and S. chinensis C I, C II, C III, CIV, C V, C VI, C VII: Cytoplasm or nucleus. Ml, MII: Mitochondria. ER: Endoplasmic reticulum. P: Plastid. Po: Peroxisome
重复基因对 Repeated gene pairs | Ka | Ks | Ka/Ks |
---|---|---|---|
ScHSP20-2 vs ScHSP20-17 | 0.383 89 | 2.622 38 | 0.146 39 |
ScHSP20-5 vs ScHSP20-6 | 0.003 77 | 0.027 34 | 0.137 86 |
ScHSP20-17 vs ScHSP20-24 | 0.137 24 | 1.495 32 | 0.091 78 |
Table 3 Ka/Ks values of fragment repeat gene pairs in ScHSP20 family members
重复基因对 Repeated gene pairs | Ka | Ks | Ka/Ks |
---|---|---|---|
ScHSP20-2 vs ScHSP20-17 | 0.383 89 | 2.622 38 | 0.146 39 |
ScHSP20-5 vs ScHSP20-6 | 0.003 77 | 0.027 34 | 0.137 86 |
ScHSP20-17 vs ScHSP20-24 | 0.137 24 | 1.495 32 | 0.091 78 |
Fig. 5 Expressions of ScHSP20 gene in different tissues and under different stresses A: Leaves and fruits of Simondwood. B: Male flowers at different flowering stages. C: Different flowering stages of female flowers in Simondmu. D: Different stages and parts of seed development in Simondwood(1: Early developmental seeds. 2: Mid stage developmental seeds. 3: Late developmental seeds. 4: Dry seeds. 5: Middle stage development of seed cotyledons. 6: Mid developmental seed hypocotyl. 7: Mid stage development of seed coat). E: The expression of ScHSP20 gene under osmotic(OS), high temperature(HT), and low temperature(LT)stress
Fig. 9 Effects of heterologous expression of ScHSP20-17 gene on the tolerance of Escherichia coli to high temperature stress A: rowth curves of recombinant bacteria and control bacteria under high temperature stress. B: Growth of control and recombinant bacteria before and after treatment.C: Statistics of survival rate of control bacteria and recombinant bacteria before and after high temperature stress treatment. * indicate significant differences at P<0.05
Fig. 11 Effects of heterologous expression of ScHSP20-17 gene on the tolerance of tobacco to high temperature stress A: Image of tobacco without injection of bacterial liquid under fluorescent protein lamp irradiation. B: Image of tobacco with injection of bacterial liquid under fluorescent protein lamp irradiation. C: Wild type tobacco(WT), tobacco with injection of empty bacterial liquid(PRI), and tobacco leaves with injection of bacterial liquid carrying PRI-ScHSP20-17-GFP vector qRT PCR analysis. D-E: Wild type tobacco. F: Tobacco with instantaneous rotation of empty liquid(PRI). G: Tobacco with instantaneous rotation of ScHSP20-17. H: MDA content determination
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