Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (6): 171-180.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1341
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MENG Guo-qiang(), GUAN Jian-wen, NIU Chun-mei, ZHOU Ying, SHEN Su-lin, WEI You-heng()
Received:
2022-11-01
Online:
2023-06-26
Published:
2023-07-07
Contact:
WEI You-heng
E-mail:m17854338552@163.com;yhwei@yzu.edu.cn
MENG Guo-qiang, GUAN Jian-wen, NIU Chun-mei, ZHOU Ying, SHEN Su-lin, WEI You-heng. Construction and Functional Study of RagA Transgenic Drosophila[J]. Biotechnology Bulletin, 2023, 39(6): 171-180.
引物名称 Primer name | 序列 Sequence(5'-3') |
---|---|
RagA-F | AGTCACTATGGCGGCCGCCATGAAGAAAAAGGTGTTAC |
RagA-R | GATGCGGCCTCCACCGCGGCAATGGTACCTTTGGCCATG |
M-F | ATCAGAAGCAACACTTGTAAACATAAGGAACGCTC |
M-R | TTTACAAGTGTTGCTTCTGATGGCAGCGTGGGATCCG |
Q61L-F | ACTGTGGCGGTCTGGAGGGCTTC |
Q61L-R | GAAGCCCTCCAGACCGCCACAGT |
T16N-F | CCGGAAAGAACAGCATGCGCTC |
T16N-R | GAGCGCATGCTGTTCTTTCCGG |
RagA-qPCR-F | GCCAGAGCAAGAAGAACC |
RagA-qPCR-R | CAATGAAAGCGGCAAAT |
rp49-qPCR-F | GCCGCTTCAAGGGACAGT |
rp49-qPCR-R | CGATCTCGCCGCAGTAAA |
Table 1 PCR primer sequences
引物名称 Primer name | 序列 Sequence(5'-3') |
---|---|
RagA-F | AGTCACTATGGCGGCCGCCATGAAGAAAAAGGTGTTAC |
RagA-R | GATGCGGCCTCCACCGCGGCAATGGTACCTTTGGCCATG |
M-F | ATCAGAAGCAACACTTGTAAACATAAGGAACGCTC |
M-R | TTTACAAGTGTTGCTTCTGATGGCAGCGTGGGATCCG |
Q61L-F | ACTGTGGCGGTCTGGAGGGCTTC |
Q61L-R | GAAGCCCTCCAGACCGCCACAGT |
T16N-F | CCGGAAAGAACAGCATGCGCTC |
T16N-R | GAGCGCATGCTGTTCTTTCCGG |
RagA-qPCR-F | GCCAGAGCAAGAAGAACC |
RagA-qPCR-R | CAATGAAAGCGGCAAAT |
rp49-qPCR-F | GCCGCTTCAAGGGACAGT |
rp49-qPCR-R | CGATCTCGCCGCAGTAAA |
Fig. 1 Construction of recombinant plasmid A: DNA sequencing comparison chart of pUASp-RagA-wt recombinant plasmid; B: DNA sequencing comparison chart of pUASp-RagA-Q61L recombinant plasmid; C: DNA sequencing comparison chart of pUASp-RagA-T16N recombinant plasmid
Fig. 3 qPCR detection of RagA gene expression mCherry RNAi is the control group; RagA RNAi is RagA RNA interference; RagA-wt; RagA RNAi is an overexpression of wild-type RagA based on RagA RNA interference; RagA-Q61L; RagA RNAi is RagA overexpressing GTP-bound state on the basis of RagA RNA interference; RagA-T16N; RagA RNAi is RagA overexpressing GDP-bound state on the basis of RagA RNA interference; ***: P<0.000 5; ****: P<0.000 1. The same below
基因型 Genotype | 羽化数 Eclosion number | 实际比例 Actual prop- ortions/% | 理论比例 Theoretical proportions/% |
---|---|---|---|
TM6/UAS-RagA RNAi | 897 | 100 | 50 |
Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 50 |
总数 Total | 897 | 100 | 100 |
Table 2 RagA knockdown on the statistical results of Drosophila eclosion rate
基因型 Genotype | 羽化数 Eclosion number | 实际比例 Actual prop- ortions/% | 理论比例 Theoretical proportions/% |
---|---|---|---|
TM6/UAS-RagA RNAi | 897 | 100 | 50 |
Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 50 |
总数 Total | 897 | 100 | 100 |
Fig. 4 Effects of RagA knockdown on Drosophila at different developmental stages A: Ratios from larval to pupal stages in control and RagA knockdown drosophilas. B: Eclosion rate from pupal stage to adult in control and RagA knockdown drosophilas. Error bars represent the standard deviation of 10 independent experiments. C: Developmental map of RagA knockdown and control flies. n refers to the total number of statistics. NS: No significant difference
Fig. 5 Somatic cell cloning assay for cell size A-A’’: RagA RNAi somatic cell clone; B-B’’: RagA-Q61L; RagA RNAi somatic cell clone; C-C’’: RagA-T16N; RagA RNAi somatic cell clone; D: RagA RNAi clone cell size statistics; E: RagA-Q61L; RagA RNAi clone cell size statistics; F: RagA-T16N; RagA RNAi clone cell size statistics
基因型 Genotype | 羽化数 Eclosion number | 实际比例 Actual pro- portion/% | 理论比例 Theoretical proportion/% |
---|---|---|---|
UAS-RagA-wt /+; Tub-Gal4/UAS-RagA RNAi | 214 | 14.8 | 12.5 |
UAS-RagA-wt /+; Tub-Gal4/MKRS | 220 | 15.3 | 12.5 |
UAS-RagA-wt /+; MKRS/TM6 | 204 | 14.1 | 12.5 |
UAS-RagA-wt /+; UAS-RagA RNAi/TM6 | 199 | 13.8 | 12.5 |
SM6/+; Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 12.5 |
SM6/+; Tub-Gal4/MKRS | 192 | 13.4 | 12.5 |
SM6/+; MKRS/TM6 | 212 | 14.7 | 12.5 |
SM6/+; UAS-RagA RNAi/TM6 | 201 | 13.9 | 12.5 |
总数 Total | 1 442 | 100 | 100 |
Table 3 Statistical results of the lethal effect of RagA overexpression on Drosophila
基因型 Genotype | 羽化数 Eclosion number | 实际比例 Actual pro- portion/% | 理论比例 Theoretical proportion/% |
---|---|---|---|
UAS-RagA-wt /+; Tub-Gal4/UAS-RagA RNAi | 214 | 14.8 | 12.5 |
UAS-RagA-wt /+; Tub-Gal4/MKRS | 220 | 15.3 | 12.5 |
UAS-RagA-wt /+; MKRS/TM6 | 204 | 14.1 | 12.5 |
UAS-RagA-wt /+; UAS-RagA RNAi/TM6 | 199 | 13.8 | 12.5 |
SM6/+; Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 12.5 |
SM6/+; Tub-Gal4/MKRS | 192 | 13.4 | 12.5 |
SM6/+; MKRS/TM6 | 212 | 14.7 | 12.5 |
SM6/+; UAS-RagA RNAi/TM6 | 201 | 13.9 | 12.5 |
总数 Total | 1 442 | 100 | 100 |
基因型 Genotype | 羽化数 Eclosion number | 实际比例 Actual pro- portions/% | 理论比例 Theoretical proportions/% |
---|---|---|---|
UAS-RagA-Q61L/+; Tub-Gal4/UAS-RagA RNAi | 63 | 4.2 | 12.5 |
UAS-RagA-Q61L/+; Tub-Gal4/MKRS | 251 | 16.6 | 12.5 |
UAS-RagA-Q61L/+; MKRS/TM6 | 253 | 16.7 | 12.5 |
UAS-RagA-Q61L/+; UAS-RagA RNAi/TM6 | 230 | 15.2 | 12.5 |
SM6/+; Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 12.5 |
SM6/+; Tub-Gal4/MKRS | 247 | 16.3 | 12.5 |
SM6/+; MKRS/TM6 | 224 | 14.9 | 12.5 |
SM6/+; UAS-RagA RNAi/TM6 | 244 | 16.1 | 12.5 |
总数 Total | 1 512 | 100 | 100 |
Table 4 Statistical results of the lethal effect of RagA and GTP binding on Drosophila
基因型 Genotype | 羽化数 Eclosion number | 实际比例 Actual pro- portions/% | 理论比例 Theoretical proportions/% |
---|---|---|---|
UAS-RagA-Q61L/+; Tub-Gal4/UAS-RagA RNAi | 63 | 4.2 | 12.5 |
UAS-RagA-Q61L/+; Tub-Gal4/MKRS | 251 | 16.6 | 12.5 |
UAS-RagA-Q61L/+; MKRS/TM6 | 253 | 16.7 | 12.5 |
UAS-RagA-Q61L/+; UAS-RagA RNAi/TM6 | 230 | 15.2 | 12.5 |
SM6/+; Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 12.5 |
SM6/+; Tub-Gal4/MKRS | 247 | 16.3 | 12.5 |
SM6/+; MKRS/TM6 | 224 | 14.9 | 12.5 |
SM6/+; UAS-RagA RNAi/TM6 | 244 | 16.1 | 12.5 |
总数 Total | 1 512 | 100 | 100 |
基因型 Genotype | 羽化数 Eclosion number | 实际比例 Actual pro- portion/% | 理论比例 Theoretical proportion/% |
---|---|---|---|
UAS-RagA-T16N/+; Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 12.5 |
UAS-RagA-T16N/+; Tub-Gal4/MKRS | 240 | 17.2 | 12.5 |
UAS-RagA-T16N/+; MKRS/TM6 | 228 | 16.3 | 12.5 |
UAS-RagA-T16N/+; UAS-RagA RNAi/TM6 | 217 | 15.5 | 12.5 |
SM6/+; Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 12.5 |
SM6/+; Tub-Gal4/MKRS | 248 | 17.8 | 12.5 |
SM6/+; MKRS/TM6 | 235 | 16.8 | 12.5 |
SM6/+; UAS-RagA RNAi/TM6 | 229 | 16.4 | 12.5 |
总数 Total | 1 397 | 100 | 100 |
Table 5 Statistical results of the lethal effect of RagA and GDP binding on Drosophila
基因型 Genotype | 羽化数 Eclosion number | 实际比例 Actual pro- portion/% | 理论比例 Theoretical proportion/% |
---|---|---|---|
UAS-RagA-T16N/+; Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 12.5 |
UAS-RagA-T16N/+; Tub-Gal4/MKRS | 240 | 17.2 | 12.5 |
UAS-RagA-T16N/+; MKRS/TM6 | 228 | 16.3 | 12.5 |
UAS-RagA-T16N/+; UAS-RagA RNAi/TM6 | 217 | 15.5 | 12.5 |
SM6/+; Tub-Gal4/UAS-RagA RNAi | 0 | 0 | 12.5 |
SM6/+; Tub-Gal4/MKRS | 248 | 17.8 | 12.5 |
SM6/+; MKRS/TM6 | 235 | 16.8 | 12.5 |
SM6/+; UAS-RagA RNAi/TM6 | 229 | 16.4 | 12.5 |
总数 Total | 1 397 | 100 | 100 |
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