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功能丧失突变透示ATS1对拟南芥种子发育的非必需作用
doi: 10.11833/j.issn.2095-0756.20220738
Loss-of-function mutations in ATS1 reveal its dispensable role in normal seed development of Arabidopsis
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摘要:
目的 甘油脂是生物膜的重要组成成分，植物中的ATS1催化甘油脂原核合成途径的第1步酰化反应，但目前ATS1在植物正常生长发育中的功能并不完全清楚。本研究目的是运用反向遗传学手段剖析ATS1功能丧失对植物生长发育的影响。 方法 运用CRISPR/Cas9基因编辑技术，构建拟南芥Arabidopsis ATS1基因功能丧失型突变体，并比较分析突变体与野生型在整个生育期的表型差异。 结果 分子鉴定显示：多个突变体中ATS1基因的第1个外显子碱基数呈非3的倍数的缺失或插入，从而导致移码突变或翻译提前终止。这些突变体的叶片中多不饱和脂肪酸C16:3的含量急剧下降，而C18:3 含量则显著增加。相随的表型分析显示：ATS1基因功能丧失有时会使叶片略显黄色，但对种子发育未产生可见影响。 结论 在正常生长条件下，ATS1并非拟南芥种子发育所必需的。图3表2参25 Abstract:Objective Glycerolipids are the main constituents of biological membranes. ATS1 catalyzes the first acylation reaction in the prokaryotic pathway of glycerolipid synthesis. However, the function of ATS1 in normal plant growth and development is not completely understood. The present study was intended to dissect the effect of loss of function of ATS1 on plant growth and development by taking a reverse genetic approach. Method Loss-of-function mutants of the ATS1 gene were constructed by using CRISPR/Cas9 gene editing technology. Then, comparative analysis was conducted on phenotypic difference between the mutants and wild type Arabidopsis during the entire growth phase. Result Molecular characterization of multiple mutants revealed that the number of base pairs inserted or deleted in the first exon of the ATS1 gene is not a multiple of three, resulting in frameshift mutations or premature translation termination. Consistent with this, the content of polyunsaturated fatty acid 16:3 in the leaves of these mutants decreased sharply, concomitant with significant increases in the content of 18:3. Meanwhile, phenotypic analysis showed that loss of ATS1 gene function sometimes made the leaves turn slightly yellow, while having no visible effect on seed development. Conclusion The above results strongly indicate that ATS1 is dispensable for Arabidopsis seed development under normal growth conditions. [Ch, 3 fig. 2 tab. 25 ref.] -
Key words:
- Arabidopsis /
- ATS1 /
- gene editing /
- ats1 mutant
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图 1 转基因拟南芥的mCherry荧光蛋白鉴定与ATS1编辑产物的聚丙烯酰胺凝胶电泳鉴定
Figure 1 Screening of transgenic plants carrying the mCherry fluorescent protein and those with CRISPR/Cas9-edited ATS1 gene product
图 2 不同ats1突变体中ATS1基因的突变位点序列
Figure 2 Sequences of mutational sites in ATS1 gene in different ats1 mutants
图 3 营养生长与生殖生长阶段野生型拟南芥与ats1突变体的表型比较
Figure 3 Phenotypic comparison of the wild type and ats1 mutants during the vegetative and reproductive stages
表 1 不同ats1突变体名称及其相应突变位点序列信息
Table 1. Designation of different ats1 mutants and the sequences of corresponding mutational sites
突变体 突变位点 ats1-1 92~314 bp：215 bp缺失；8 bp替换 ats1-2 91~92 bp：插入1 bp；293~294 bp：插入1 bp ats1-3 91~92 bp：7 bp插入；275~289 bp：11 bp缺失，
4 bp替换
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表 2 野生型拟南芥与ats1突变体叶片的脂肪酸组分
Table 2. Leaf fatty acid composition of ats1 mutants and wild-type Arabidopsis
脂肪酸 脂肪酸组分含量/% C16:0 C16:1 C16:3 C18:0 C18:1 C18:2 C18:3 WT 14.91±0.73 a 7.35±0.53 a 11.56±0.38 a 6.17±1.55 a 4.37±0.59 b 14.89±1.30 b 38.50±3.04 b ats1-1 11.91±0.65 b 5.55±0.69 b 0.70±0.15 b 3.89±0.87 a 8.65±0.75 a 18.61±0.54 a 49.14±2.24 a ats1-2 11.20±0.18 b 5.93±0.89 ab 0.65±0.15 b 4.67±0.32 a 8.89±1.06 a 18.67±0.98 a 48.31±1.68 a ats1-3 12.29±0.81 b 6.00±0.93 ab 0.57±0.18 b 6.02±1.62 a 9.08±1.02 a 18.28±0.88 a 46.04±1.45 a 说明：WT为野生型对照，n=3，不同小写字母表示不同株系间显著差异(P＜0.05)
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