RagA转基因果蝇的构建及功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-1341

摘要/Abstract

摘要：

Rag GTPase属于Ras家族成员的GTP结合蛋白，定位于溶酶体。果蝇RagA蛋白是哺乳动物RagA/RagB蛋白同源物，在果蝇中RagA与RagC结合，共同调节TORC1活性。本实验室发现敲减RagA使果蝇发育停滞在蛹期。为了探究具体原因，基于密码子的简并性，通过融合PCR、同源重组克隆的方法构建了改变RagA RNA干扰靶标位点的过表达载体pUASp-RagA-wt；在此基础上，通过点突变的方式构建了与GTP结合的RagA过表达载体pUASp-RagA-Q61L和与GDP结合的RagA过表达载体pUASp-RagA-T16N。通过显微注射和遗传杂交等方式分别筛选出pUASp-RagA-wt、pUASp-RagA-Q61L、pUASp-RagA-T16N转基因果蝇。通过体细胞克隆的方法检测了不同活性状态的RagA对TORC1活性的影响，RagA调节TORC1活性：与GTP结合处于激活态；与GDP结合处于失活态。为确定GTP/GDP状态下RagA活性对果蝇发育的影响，将UASp转基因系果蝇与Tub-Gal4/TM6果蝇杂交并统计后代羽化率，判断由GTP/GDP状态控制的RagA活性对果蝇生长发育的影响。结果发现，RagA敲减果蝇由于蛹期阶段内出现障碍不能发育为成虫。在RagA敲减的背景下，过表达野生型RagA能够完全挽救RagA敲减所导致的果蝇致死。过表达GTP结合型的RagA-Q61L能够部分挽救RagA敲减的致死效应，过表达GDP结合型的RagA-T16N不能挽救RagA敲减的致死效应。表明RagA在果蝇生长发育过程中起着重要的作用，且RagA与GTP/GDP结合需保持动态平衡。RagA仅与GTP或GDP结合会使TORC1活性处于过高或过低的状态，影响果蝇的生长发育。

关键词: RagA, 显微注射, 果蝇

Abstract:

Rag GTPase is GTP-binding proteins belonging to a member of the Ras family and localizes on lysosomes. Drosophila RagA protein, a homologue of mammalian RagA/RagB protein, forms a complex with RagC to regulate TORC1 activity. Here we found that knockdown of RagA caused Drosophila development to stagnate at the pupal stage. To explore the function of RagA on Drosophila development, we constructed the overexpressing vector pUASp-RagA-wt with RagA RNA interfering target sites changed. On this basis, the GTP-binding RagA-overexpressed plasmid pUASp-RagA-Q61L and the GDP-binding RagA-overexpressed plasmid pUASp-RagA-T16N were constructed by point mutation. The pUASp-RagA-wt, pUASp-RagA-Q61L and pUASp-RagA-T16N plasmids were screened by microinject and screentransgene lines. The effects of different forms of RagA on the TORC1 activity were evaluated by the method of somatic cell cloning, RagA regulated the TORC1 activity, and it was in the activated state after binding with GTP, and inactivated after binding with GDP. To determine the effects of RagA activity on the Drosophila development under GTP/GDP state, the UASp transgene lines were crossed with Tub-Gal4/TM6 and the eclosion rate of offspring was counted. The results showed that Drosophila with RagA knockdown failed to develop into adults due to obstacles in the pupal stage. The overexpression of wild-type RagA completely avoided the death of Drosophila caused by RagA knockdown, confirming that depletion of RagA was the cause of developmental defects in RagA RNAi. The overexpression of GTP-bound RagA-Q61L partially reduced the lethal effect of Drosophila with RagA knockdown, while the overexpression of GDP-bound RagA-T16N could not decrease the lethal effect of Drosophila with RagA knockdown at all. Our work suggests that RagA plays an important role in the growth and development, and the binding of RagA and GTP/GDP needs to be in a dynamic equilibrium. Binding of RagA only to GTP or GDP would make TORC1 activity too high or too low, thus affecting fruit fly growth and development.

Key words: RagA, microinjection, Drosophila

孟国强, 管建文, 牛春梅, 周颖, 沈苏林, 韦有恒. RagA转基因果蝇的构建及功能研究[J]. 生物技术通报, 2023, 39(6): 171-180.

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.

图/表 10

表1 PCR引物序列

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

图1 重组质粒的构建 A：pUASp-RagA-wt重组质粒DNA测序对比图；B：pUASp-RagA-Q61L重组质粒DNA测序对比图；C：pUASp-RagA-T16N重组质粒DNA测序对比图

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

图2 果蝇筛选杂交示意图

Fig. 2 Schematic diagram of Drosophila screening hybrid

图3 qPCR检测RagA基因的表达 mCherry RNAi为对照组；RagA RNAi为RagA RNA干扰；RagA-wt; RagA RNAi为RagA RNA干扰的基础上过表达野生型RagA；RagA-Q61L; RagA RNAi为RagA RNA干扰的基础上过表达GTP结合状态的RagA；RagA-T16N; RagA RNAi为RagA RNA干扰的基础上过表达GDP结合状态的RagA；***: P<0.000 5; ****: P<0.000 1。下同

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

表2 RagA敲减对果蝇羽化率的统计结果

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

图4 RagA敲减对果蝇不同发育阶段的影响 A：对照组和RagA敲减果蝇中从幼虫到蛹期的比率；B：对照组和RagA敲减果蝇从蛹期到成虫的羽化率；误差条代表10个独立实验的标准偏差； C：RagA敲减和对照果蝇的发育图。n表示统计的总数。NS：无显著差异

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

图5 体细胞克隆检测细胞大小 A-A’’：RagA RNAi体细胞克隆； B-B’’：RagA-Q61L; RagA RNAi体细胞克隆； C-C’’：RagA-T16N; RagA RNAi体细胞克隆；D：RagA RNAi克隆细胞大小统计；E：RagA-Q61L; RagA RNAi克隆细胞大小统计；F：RagA-T16N; RagA RNAi克隆细胞大小统计

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

表3 过表达RagA对果蝇致死效应的统计结果

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

表4 RagA与GTP结合状态对果蝇致死效应统计结果

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

表5 RagA与GDP结合状态对果蝇致死效应的统计结果

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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