Heterologous overexpression of MtATG7 gene from Medicago truncatula promotes resistance to carbon and nitrogen starvation in Arabidopsis thaliana
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摘要:目的
真核生物通过保守的自噬途径降解错误折叠的蛋白质或受损细胞器，实现对营养物质的循环再利用。本文旨在解析苜蓿自噬基因在植物应对营养胁迫中发挥的功能，为指导苜蓿的选育和改良提供参考。
方法以自噬途径的关键限速基因ATG7(Autophagy-related gene 7)为切入点，分析ATG7氨基酸序列在不同植物中的相似性。利用蒺藜苜蓿Medicago truncatula的MtATG7基因过表达载体转化拟南芥植株，获得异源过表达植株35S::MtATG7和互补拟南芥突变体的atg7/35S::MtATG7植株，并对其抗胁迫表型和自噬活性进行分析。
结果在碳饥饿条件下，atg7/35S::MtATG7能够挽救atg7突变体叶片早衰的表型；恢复到正常生长条件以后，35S::MtATG7和atg7/35S::MtATG7植株存活率和野生型相比都显著提高。GFP-ATG8e的剪切试验表明，atg7/35S::MtATG7能够恢复atg7突变体的自噬降解活性。在氮饥饿条件下过表达MtATG7同样能够减缓拟南芥叶片的衰老速度。
结论异源过表达MtATG7能增强拟南芥碳氮饥饿抗性的生物学功能，本研究为进一步利用MtATG7基因改良苜蓿农艺性状提供了理论依据。
Abstract:ObjectiveEukaryotes use autophagy pathway to recycle nutrients by degrading misfolded proteins or damaged organelles. This study aims to analyze the function of Medicago autophagy genes in plant response to nutritional stress, and provide a reference for the breeding and improvement of Medicago.
MethodWe focused on the ATG7 gene (Autophagy-related gene 7), which serves as the rate-limiting gene in the autophagy pathway, and investigated the similarities of ATG7 amino acid sequences among different plant species. The overexpression vector of Medicago truncatula MtATG7 gene was transformed to Arabidopsis thaliana, to generate the heterologous overexpression lines 35S::MtATG7 and the complementary lines atg7/35S::MtATG7. The plant resistance to stress and autophagic activity were analyzed.
ResultUnder carbon starvation, atg7/35S::MtATG7 rescued the phenotype of premature leaf senescence of atg7 mutant, and the survival rates of 35S::MtATG7 and atg7/35S::MtATG7 plants were significantly higher than that of wildtype. GFP-ATG8e cleavage assay suggested that atg7/35S::MtATG7 restored the deficiency of autophagic degradation activity in atg7 mutants. Under nitrogen starvation, overexpression of MtATG7 also slowed down the senescence rate of A. thaliana leaves.
ConclusionHeterologous overexpression of MtATG7 can enhance the resistance of A. thaliana under carbon and nitrogen starvation, which provides a theoretical basis for further improving Medicago agronomic traits by using MtATG7 gene.
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图 1 不同植物ATG7基因的系统发育树分析和保守结构域预测
Figure 1. Phylogenetic tree analysis and domain prediction of ATG7 gene from different species
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图 2 MtATG7过表达载体的构建
A：苜蓿MtATG7基因的克隆；B：35S::MtATG7过表达载体的菌落PCR结果，M：Trans2K Plus II DNA Marker，1~10：菌落；目标条带大小为2097 bp左右
Figure 2. Construction of MtATG7 overexpression vector
A: Cloning of MtATG7 gene in Medicago; B: Colony PCR results of 35S::MtATG7 overexpression vector, M: Trans2KPlus II DNA Marker, 1−10: Colony; The target band size is about 2097 bp
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图 3 MtATG7促进转基因拟南芥抵抗碳胁迫
A：将苗龄11 d的野生型Col、自噬突变体atg7以及转基因atg7/MtATG7#1、atg7/MtATG#2、MtATG7-OX#1、MtATG7-OX#2拟南芥幼苗移到不含糖的1/2MS培养基中，黑暗条件下培养9 d后的表型；B：在碳胁迫9 d后分别对每个株系取2株幼苗拍照；比例尺=5 mm
Figure 3. MtATG7 promotes resistance to carbon starvation in transgenic Arabidopsis
A: Phenotypes for 11-day-old wild-type Col, autophagy mutant atg7, and transgenic atg7/MtATG7#1, atg7/MtATG#2, MtATG7-OX#1, MtATG7-OX#2 Arabidopsis seedlings after transferred to sugar-free 1/2MS medium and cultured under dark conditions for 9 days; B: After 9 days of carbon starvation stress, two seedlings from each line were photographed; Scale bar = 5 mm
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图 4 碳胁迫前后的拟南芥幼苗叶绿素含量
数据为平均值±标准差, n=8；相同处理柱子上方的不同小写字母表示各株系之间差异显著(P<0.05，LSD法)
Figure 4. Chlorophyll content of Arabidopsis seedling before and after carbon stress
Data are means ± SDs, n=8; Different lowercase letters on bars of the same treatment indicate significant differences among strains (P<0.05，LSD test)
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图 5 MtATG7促进转基因拟南芥在碳胁迫下的生存
图中为将苗龄3周的野生型Col、自噬突变体atg7以及转基因atg7/MtATG7#1、atg7/MtATG#2、MtATG7-OX#1、MtATG7-OX#2拟南芥植株黑暗处理7 d，恢复光照8 d后的表型；比例尺=1 cm
Figure 5. MtATG7 promotes survival of transgenic Arabidopsis under carbon starvation
3-week-old Arabidopsis seedlings of wild type Col, autophagy mutant atg7, and transgenic lines atg7/MtATG7#1, atg7/MtATG#2, MtATG7-OX#1, and MtATG7-OX#2 were treated in the dark for 7 days，and the phenotypes after 8 days of recovery under light are shown in the figure; Scale bar = 1 cm
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图 6 碳胁迫后的拟南芥幼苗的存活率
统计各株系在恢复光照8 d后的存活率；柱子上方的不同小写字母表示各株系之间差异显著(P<0.05，LSD法)
Figure 6. Survival of Arabidopsis seedlings after carbon stress
The survival rate of each strain after 8 days of light recovery are calculated; Different lowercase letters on bars indicate significant differences among strains (P<0.05，LSD test)
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图 7 MtATG7使拟南芥atg7突变体的自噬活性恢复
免疫印迹试验检测拟南芥GFP-ATG8e、atg7-3/GFP-ATG8e以及转基因MtATG7/atg7-3/GFP-ATG8e的GFP剪切活性；上样量对照为丽春红染色的Rubisco蛋白
Figure 7. MtATG7 restores autophagy activity of atg7 mutant in Arabidopsis
Western blot was used to detect the GFP cleavage activities of GFP-ATG8e, atg7-3/ ATG8e and MtATG7/atg7-3/ATG8e; Rubisco protein with ponceau staining was used as the loading control
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图 8 MtATG7促进转基因拟南芥抵抗氮胁迫
A：将苗龄11 d的野生型Col、自噬突变体atg7以及转基因atg7/MtATG7#1、atg7/MtATG#2、MtATG7-OX#1、MtATG7-OX#2拟南芥幼苗移入缺氮的1/2MS培养基中培养7 d后的表型；B：在氮胁迫7 d后分别对每个株系取2株幼苗拍照；比例尺=5 mm
Figure 8. MtATG7 promotes resistance to nitrogen starvation in transgenic Arabidopsis
A: Phenotypes for 11-day-old wild-type Col, autophagy mutant atg7, and transgenic atg7/MtATG7#1, atg7/MtATG#2, MtATG7-OX#1, MtATG7-OX#2 Arabidopsis seedlings after transferred to nitrogen-deficient 1/2MS medium and cultured for 7 days; B: After 7 days of nitrogen starvation stress, two seedlings from each line were photographed; Scale bar = 5 mm
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图 9 氮胁迫前后的拟南芥幼苗叶绿素含量
数据为平均值±标准差, n=8；相同处理柱子上方的不同小写字母表示各株系之间差异显著(P<0.05，LSD法)
Figure 9. Chlorophyll content of Arabidopsis seedlings before and after N stress
Data are means ± SDs, n=8; Different lowercase letters on bars of the same treatment indicate significant differences among strains (P<0.05，LSD test)
表 1 PCR特异性引物序列
Table 1 Specific primers used for PCR
引物类型
Primer type引物名称
Primer name引物序列(5′→3′)
Primer sequence目的基因引物
Target gene primerMtATG7-F gaacacgggggactGGATCCATGGCTTTGCTCCAATTTAT MtATG7-R AATGTTTGAACGATCTGCAGTCATATTTCAAAACAATCTT 自噬突变体鉴定引物
Autophagy mutant identification primeratg7-F TCTCTTGTTGGTCAAGCCTC atg7-R CATTGTTGGTCTAGAGTTCG LB1 GCCTTTTCAGAAATGGATAAATAGCCTTGCTT 
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