紫花苜蓿甘氨酸脱羧酶H-蛋白基因MsGDC-H1功能分析

doi:10.11686/cyxb2021477

摘要/Abstract

摘要：

光呼吸通过清除2-磷酸乙醇酸（2-PG）使氧合光合作用成为可能，该过程对C₃植物至关重要。H-蛋白是光呼吸过程中将甘氨酸转化为丝氨酸的甘氨酸脱羧酶（GDC）的关键组成蛋白之一。本研究克隆了紫花苜蓿MsGDC-H1，该基因编码166个氨基酸，具有1个硫辛酰基附着位点保守结构域和1个N6-硫辛酰赖氨酸保守位点。进化分析表明，MsGDC-H1蛋白与双子叶植物的甘氨酸脱羧酶H-蛋白（GDC-H）亲缘关系近。表达模式分析表明，MsGDC-H1在苜蓿叶中表达丰度高，且受光诱导。为了探究MsGDC-H1基因对拟南芥生长的影响，分别使用光诱导的茎叶特异性启动子ST-LS1和组成型启动子CaMV 35S驱动MsGDC-H1（ST-LS1：：MsGDC-H1； CaMV 35S：：MsGDC-H1）在拟南芥中异源表达。检测过表达植株生物量、淀粉、可溶性糖含量以及光合速率。数据分析显示，CaMV 35S：：MsGDC-H1过表达拟南芥（G系列植株）生长受阻，淀粉含量比ST-LS1：：MsGDC-H1特异性表达拟南芥（GS系列植株）增加了34%~67%，比野生型（WT）增加了7.3%~33.7%；可溶性糖含量比GS系列降低了36%~38%，比WT增加了44.3%~49.7%；与WT相比，GS系列植株生长更快，淀粉含量无显著差异（P>0.05），可溶性糖含量显著增加（P<0.05）。试验结果表明，MsGDC-H1基因在调控拟南芥光合速率、碳水化合物合成以及生长等方面发挥重要作用，未来可作为提高苜蓿产量的基因工程育种候选基因。

关键词: 甘氨酸脱羧酶H-蛋白, 产量, 光合速率, 糖含量, 拟南芥

Abstract:

Photorespiration is essential for C₃ plants， allowing oxygenated photosynthesis by removing 2-phosphoglycolate. H-protein is one of the key components of glycine decarboxylase （GDC）， which converts glycine to serine during photorespiration. The MsGDC-H1 gene was cloned from Medicago sativa. The cloned gene encoded 166 amino acids and had a conserved lipoyl-binding domain and a conserved N6-lipoyllysine site. Phylogenetic analysis showed that MsGDC-H1 was closely related to glycine decarboxylase H-protein （GDC-H） in other dicotyledons. The expression pattern analysis showed that MsGDC-H1 was highly expressed in leaves and was induced by light. To explore the effects of MsGDC-H1 on Arabidopsis thaliana growth， photoinduced stem-leaf specific promoter ST-LS1 and constitutive promoter CaMV 35S were used to drive MsGDC-H1 expression （ST-LS1：：MsGDC-H1； CaMV 35S：： MsGDC-H1）. MsGDC-H1 is heterogeneously expressed in A. thaliana. Biomass， starch and soluble sugar content and photosynthetic rate of overexpressed plants were evaluated. Data analysis showed that CaMV 35S：：MsGDC-H1 overexpression （G series） inhibited A. thaliana growth. The starch content of G series was increased by 34%-67% compared with A. thaliana in which ST-LS1：：MsGDC-H1 was overexpressed （GS series）， and was increased by 7.3%-33.7% compared with wild type （WT） A. thaliana； The soluble sugar content was decreased by 36%-38% compared with GS series and increased by 44.3%-49.7% compared with WT A. thaliana. Compared with WT， GS series plants grew faster， starch content was not significantly different （P>0.05）， and soluble sugar content was significantly increased （P<0.05）. The experiment results show that MsGDC-H1 plays an important role in regulating photosynthetic rate， carbohydrate synthesis and growth of A. thaliana， and can be used as a candidate gene in genetic engineering breeding to improve alfalfa yield in the future.

Key words: glycine decarboxylase H-protein, yield, photosynthetic rate, carbohydrate content, Arabidopsis thaliana

尉春雪, 何飞, 许蕾, 李霄, 张立霞, 李明娜, 陈林, 康俊梅, 杨青川, 龙瑞才. 紫花苜蓿甘氨酸脱羧酶H-蛋白基因MsGDC-H1功能分析[J]. 草业学报, 2022, 31(12): 95-105.

Chun-xue WEI, Fei HE, Lei XU, Xiao LI, Li-xia ZHANG, Ming-na LI, Lin CHEN, Jun-mei KANG, Qing-chuan YANG, Rui-cai LONG. Functional analysis of glycine decarboxylase H-protein gene MsGDC-H1 in Medicago sativa[J]. Acta Prataculturae Sinica, 2022, 31(12): 95-105.

图/表 8

图1 MsGDC-H1蛋白功能及结构分析A：植物光呼吸过程中甘氨酸脱羧酶功能模式Functional model of glycine decarboxylase during photorespiration in plants； H：甘氨酸脱羧酶H-蛋白 Glycine decarboxylase H-protein； P：甘氨酸脱羧酶P-蛋白Glycine decarboxylase P-protein； T：甘氨酸脱羧酶T-蛋白Glycine decarboxylase T-protein； L：甘氨酸脱羧酶L-蛋白Glycine decarboxylase L-protein； aa：氨基酸Amino acid；甘氨酸在H-、P-、T-、L-蛋白作用下转变为丝氨酸Glycine is converted to serine by H-， P-， T-， and L-proteins；B： MsGDC-H1保守结构域分析Analysis of MsGDC-H1 conserved domain；C： MsGDC-H1蛋白三维结构预测，黄色区域为硫辛酰基附着位点保守结构域Prediction of three-dimensional structure of MsGDC-H1 protein， the yellow area is the conserved domain of lipoyl-binding.

Fig.1 Functional and structural analysis of MsGDC-H1 protein

表1 引物序列

Table 1 Primer sequence

引物Primer	引物序列Primer sequence （5′-3′）
MsH-F	ATGGCACTGAGGATGTGGGCTTC
MsH-R	TCAATGTGCAGCATCTTCTTCCTCA
ST-LS1-F	GCAAGCTTTTGTTTCTATCCACTGATGTCTG
ST-LS1-R	GCGGATCCTTGCTCTCACTACTTAGTATGA
qRT-MsH-F	CACTGAGGATGTGGGCTTCTT
qRT-MsH-R	AGCCTTCATGCTTCACCCAT
qRT-Atactin-F	CAAAAGATGGCAGATGCTGAGGAT
qRT-Atactin-R	CATGCACCAGTATGACGAGGTCG
qRT-Msactin-F	GAAATCACAGCACTTGCACC
qRT-Msactin-R	AAGCCTTTGATCTTGAGAGC

图2 多物种中H-蛋白进化树分析Ⅰ、Ⅱ：H-蛋白在高等植物中的两个亚家族Two subfamilies of H-proteins in higher plants； Ms：紫花苜蓿M. sativa；MsGDC-H1：MsG0580024678.01.T01；MsGDC-H2：MsG0480022167.01.T01；MsGDC-H3：MsG0780040690.01.T01［20］；Mt：蒺藜苜蓿M. truncatula；MtGDC-H1：XP_003611464.1；MtGDC-H2：XP_003607890.1； At：拟南芥A. thaliana；AtGDC-H1：NP_181080.1；AtGDC-H2：NP_174525.1；Nt：烟草N. tabacum；NtGDC-H1：XP_016501895.1；NtGDC-H2：XP_016454762.1；NtGDC-H3：XP_016447704.1；NtGDC-H4：XP_016484703.1；Gm：大豆G. max；GmGDC-H1：NP_001238231.1；GmGDC-H2：XP_003539169.1；GmGDC-H3：XP_003556280.1；GmGDC-H4：NP_001237081.2；GmGDC-H5：NP_001242407.1；Os：水稻O. sativa；OsGDC-H1： XP_015627588.1；OsGDC-H2：XP_015643398.1；OsGDC-H3：XP_015627588.1；Pp：小立碗藓P. patens；PpGDC-H1：XP_001780120.1；PpGDC-H2：XP_001769387.1；Cr：莱茵衣藻C. reinhardtii；CrGDC-H：XP_001696637.1；Sm：江南卷柏S. moellendorffii；SmGDC-H1：XP_002980848.1；SmGDC-H2：XP_002989355.1；La：狭叶羽扇豆L. angustifolius；LaGDC-H1：XP_019445983.1；LaGDC-H2：XP_019424562.1；LaGDC-H3：XP_019420317.1；LaGDC-H4：XP_019445859.1；Sb：高粱S. bicolor；SbGDC-H1：XP_002451618.1；SbGDC-H2：XP_002451618.1；Zm：玉米Z. mays；ZmGDC-H1：NP_001141253.1；ZmGDC-H2：Zm00001eb207290；ZmGDC-H3：NP_001141253.1；ZmGDC-H4： Zm00001eb391290；ZmGDC-H5：Zm00001eb025960；Ac：凤梨A. comosus；AcGDC-H1：XP_020109774.1；AcGDC-H2：Aco008361.1. path 1.

Fig.2 Phylogenetic tree analysis of H-protein in multiple species

表2 GDC-H在不同物种中的信息

Table 2 Information of GDC-H in different species

分类 Category	物种 Species	光合类型 Photosynthetic types	数目Number
分类 Category	物种 Species	光合类型 Photosynthetic types	GDC-H	GDC-H外显子Exons
双子叶植物 Dicot	紫花苜蓿M.sativa	C₃	3	1，3，4
	蒺藜苜蓿M. truncatula	C₃	2	3，4
	拟南芥A. thaliana	C₃	3	3，4
	大豆G. max	C₃	5	3，4
	烟草N. tabacum	C₃	4	3，4
	狭叶羽扇豆L. angustifolius	C₃	4	2，3，4
单子叶植物 Monocot	水稻O. sativa	C₃	4	4，9
	玉米Z. mays	C₄	6	3，4，5，7
	凤梨A. comosus	CAM	2	3，4
	高粱S. bicolor	C₄	2	4
低等植物 Lower plants	小立碗藓P. patens	C₃	2	1，4
	江南卷柏S. moellendorffii	C₃	2	4
	莱茵衣藻C. reinhardtii	C₃	1	5

图3 MsGDC-H1基因表达量分析A：紫花苜蓿中MsGDC-H1对光的响应分析Response analysis of MsGDC-H1 in M. sativa to light；B： MsGDC-H1在紫花苜蓿茎、叶、花中的表达量Expression levels of MsGDC-H1 in alfalfa stems， leaves and flowers；C：MsGDC-H1在过表达拟南芥根中的循环阈值Cycle threshold of MsGDC-H1 in overexpressed A. thaliana roots；循环阈值Cycle threshold：实时荧光定量PCR 循环过程中，荧光信号开始由本底进入指数增长阶段的拐点所对应的循环次数In the real-time quantitative PCR cycle， the number of cycles corresponding to the inflection point when the fluorescence signal begins to enter the exponential growth stage from the background；D：G系列植株在黑暗6 h和光照8 h后的表达量Expression levels of G series plants after 6 h darkness and 8 h light；E：GS系列植株在黑暗6 h和光照8 h后的表达量Expression levels of GS series plants after 6 h darkness and 8 h light；G42、G45、G66表示CaMV 35S：：MsGDC-H1过表达拟南芥株系，GS39、GS34、GS3表示ST-LS1：：MsGDC-H1特异性表达拟南芥株系G42， G45， G66 indicate CaMV 35S：：MsGDC-H1 overexpression A. thaliana， GS39， GS34， GS3 indicate ST-LS1：：MsGDC-H1 specific expression A.thaliana；不同小写字母表示不同时间、不同组织、不同株系存在显著差异（P<0.05），下同Different lowercase letters indicate that there are significant differences in different time， different tissues and different lines， the same below.

Fig.3 Analysis of MsGDC-H1 expression

图4 T3代转基因拟南芥表型分析A：5周龄过表达拟南芥表型Overexpression of A. thaliana phenotype at 5 weeks；B：5周龄过表达拟南芥地上部分鲜重Overexpression of A. thaliana shoots fresh weight at 5 weeks；C：5周龄过表达拟南芥地上部分干重Overexpression of A. thaliana shoots dry weight at 5 weeks；WT：野生型Col-0 Wild type Col-0.

Fig.4 Phenotypic analysis of T3 transgenic A. thaliana

图5 转基因及野生型株系叶片中糖类含量分析相对含量Relative content：过表达株系淀粉及可溶性糖含量占WT的百分比The percentage of starch and soluble sugar content of overexpressed lines in WT.

Fig.5 Analysis of carbohydrate content in leaves of transgenic and wild-type strains

图6 转基因及野生型株系光合速率检测分析相对光合速率Relative photosynthetic rate：过表达株系光合速率占WT的百分比The percentage of photosynthetic rate of overexpressed lines in WT.

Fig.6 Detection and analysis of photosynthetic rate of transgenic and wild-type strains

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