二倍体马铃薯中SCFSLF复合体的组分分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0904

摘要/Abstract

摘要：

自交不亲和（self-incompatibility,SI）广泛存在于开花植物中,是防止近交促进杂交的一种途径,可有效维持物种的基因型多样性。车前科、茄科、蔷薇科等植物中的自交不亲和类型属于典型的配子体型自交不亲和（gametophytic self-incompatibility,GSI）。在GSI植物的杂交过程中,花粉管中的SCF^SLF复合体（Skp1/Cullin1/F-box）作为E3泛素连接酶,参与了花粉对异我型S-核糖核酸酶（S-RNase）的降解过程,是维持杂交亲和的关键因子。本研究通过整理GSI植物SCF^SLF复合体的研究进展,借助同源比对、进化树及蛋白序列分析等生物信息学分析手段,对二倍体马铃薯中SCF^SLF复合体的各个组分进行了初步分析,并对各组分基因的表达量和蛋白互作情况进行分析,最终确定了马铃薯花粉管中的SCF^SLF复合体可能是由Soltu.DM.06G001040,Soltu.DM.11G004330,Soltu.DM.06G022320以及相应的SLF蛋白组成的。

关键词: SCF^SLF复合体, 配子体型自交不亲和, 马铃薯

Abstract:

Self-incompatibility (SI) exists widely in flowering plants,which is a way to prevent inbreeding and promote hybridization,and may effectively maintain the genotypic diversity of species. The type of SI in Plantaginaceae,Solanaceae,Rosaceae and other plants belongs to the typical gametophytic self-incompatibility (GSI). In the cross-pollination of GSI plants,the SCF^SLF complex (Skp1/Cullin1/F-box) in the pollen tube,as the E3 ubiquitin ligase,is involved in the degradation of S-RNase,which is the key factor to maintain the crossing compatibility. This study summarized the research progress of SCF^SLF complex in GSI plants,and preliminarily analyzed the components of SCF^SLF complex in diploid potato by homologous alignment,phylogenetic analysis and multiple protein sequence alignments. The gene expression and protein interaction of each component were analyzed,and it was preliminarily determined that the SCF^SLF complex in potato pollen tube may be composed of Soltu.DM.06G001040,Soltu.DM.11G004330,Soltu.DM.06G022320 and the corresponding SLF proteins.

Key words: SCF^SLF complex, gametophytic self-incompatibility, Solanum tuberosum

高蒙, 李富婷, 魏湛林, 张赛行, 白如仟, 尚轶, 马玲. 二倍体马铃薯中SCF^SLF复合体的组分分析[J]. 生物技术通报, 2022, 38(4): 117-125.

GAO Meng, LI Fu-ting, WEI Zhan-lin, ZHANG Sai-hang, BAI Ru-qian, SHANG Yi, MA Ling. Component Analysis of SCF^SLF Complex in Diploid Potato[J]. Biotechnology Bulletin, 2022, 38(4): 117-125.

图/表 7

图1 典型 SCF复合体模式图

Fig.1 Typical structural pattern of SCF complex

表1 马铃薯SSK1与Cullin1荧光定量引物

Table 1 Fluorescent quantitative primers for SSK1 and Cullin1 in potato（S. tuberosum）

引物名称 Primer	引物序列 Sequence（5'-3'）
4330-SSK1-F	ATGGAGTTGTGTTGCCAATC
4330-SSK1-R	AATCTCAGGACCTTCAAATGC
1040-CLU1-F	GTGTCATGTGCCAAGTACAA
1040-CLU1-R	CATAACGTCTATCCTTGCCG
QEF1a-F	GATGGTCAGACCCGTGAACATGC
QEF1a-R	ACGATTTCATCATACCTAGCCTTGGAGT

表2 二倍体马铃薯DM中SCFSLF复合体组分的同源比对

Table 2 Homologous comparison of SCFSLF complex components in diploid potato DM

	基因编号Gene ID	基因位点Location	E值E value
PiSSK1同源基因 PiSSK1-homologous gene	Soltu.DM.11G004330	chr11：4294163..4295355 reverse	1.71E-102
	Soltu.DM.11G016290	chr11：30776770..30778841 reverse	2.83E-44
	Soltu.DM.11G004300	chr11：4286144..4286630 forward	9.28E-43
	Soltu.DM.10G017580	chr10：47907512..47910644 forward	9.30E-42
	Soltu.DM.01G051200	chr01：87717008..87719187 forward	2.54E-41
	Soltu.DM.11G004500	chr11：4499656..4500133 reverse	1.32E-40
PiCUL1-P同源基因 PiCUL1-P-homologous genes	Soltu.DM.06G001040	chr06：1465504..1471494 reverse	0
	Soltu.DM.06G035050	chr06：58938032..58944924 reverse	0
	Soltu.DM.09G023090	chr09：58921771..58929345 reverse	0
	Soltu.DM.01G022080	chr01：60076281..60087621 forward	0
	Soltu.DM.09G003030	chr09：2388523..2390740 reverse	0
	Soltu.DM.09G002940	chr09：2349588..2351796 forward	0
	Soltu.DM.01G022100	chr01：60106453..60114114 forward	0
	Soltu.DM.01G022150	chr01：60164455..60171592 forward	0
	Soltu.DM.09G003010	chr09：2377567..2379367 reverse	0
	Soltu.DM.01G022120	chr01：60134989..60155075 forward	0
PiRbx1同源基因 PiRbx1-homologous genes	Soltu.DM.06G022320	chr06：48782308..48785375 reverse	1.82E-71

图2 SSK1蛋白序列与表达量分析 A：GSI植物中SSK基因的系统进化树（Pb：Pyrus bretschneideri 梨;Md：Malus domestica 苹果;Pet：Prunus tenella 李;Cr：Citrus reticulata 橘;Ah：Antirrhinum金鱼草;Pi：Petunia inflata膨大矮牵牛;Ph：Petunia hybrida杂交矮牵牛;L：Lily百合;红色三角所在分支是蔷薇科和茄科发生分离的分支）;B：GSI植物中SSK蛋白的序列比对;C：Soltu.DM.11G004330在E172不同部位中的表达量,以stamen中的表达量为对照组,利用2-∆∆Ct法进行相对基因表达量的计算

Fig. 2 SSK1 protein sequence and expression analysis A：Phylogenetic tree of SSK genes in GSI plants（The branch marked by the red triangle is the branch where Rosaceae and Solanaceae separated）. B：Sequence alignment of SSK protein in GSI plants. C：The expression of Soltu.DM.11G004330 in different organs of E172 is calculated by 2-∆∆Ct with stamen as control

图3 Cullin1蛋白进化与表达量分析 A：GSI植物中CUL基因的系统进化树（Pi：Petunia inflata 膨大矮牵牛;Ph：Petunia hybrida 杂交矮牵牛;Cr：Citrus reticulata橘;Md：Malus domestica苹果;Pet：Prunus tenella李;Pb：Pyrus bretschneideri梨;Ah：Antirrhinum金鱼草;L：Lily百合;红色三角所在分支是蔷薇科和茄科发生分离的分支）;B：Soltu.DM.06G001040在E172不同部位中的表达量,以stamen中的表达量为对照组,利用2-∆∆Ct法进行相对基因表达量的计算

Fig. 3 Cullin1 protein evolution and expression analysis A：Phylogenetic tree of CUL genes in GSI plants（The branch marked by the red triangle is the branch where Rosaceae and Solanaceae separated）. B：The expression of Soltu.DM.06G001040 in different organs of E172 is calculated by 2-∆∆Ct with stamen as control

图4 马铃薯Cullin1蛋白的多序列比对

Fig. 4 Multiple sequence alignment of Cullin1 protein in potato

图5 Soltu.DM.11G004330蛋白与SLF蛋白的互作分析 A：Soltu.DM.11G004330与SLF互作的酵母双杂交验证图（-WLHA：四缺（-Trp/W,-Leu/L,-His/H,-adenine/Ade）培养板;100,10-1,10-2分别指菌的滴定浓度;pGBKT7-53和pGADT7-T用作positive control;pGBKT7-Lam和pGADT7-T用作negative control;4330是Soltu.DM.11G004330的缩写）;B：Soltu.DM.11G004330与SLF互作的荧光素酶互补验证图（烟草叶片注射分区情况如左边所示,其中4330是Soltu.DM.11G004330的缩写）

Fig. 5 Interaction analysis of Soltu.DM.11G004330 and SLF A：Y2H of Soltu.DM.11G004330 and SLF（-WLHA,synthetic dropout media lacking tryptophan,leucine,histidine,and adenine（-Trp/W,-Leu/L,-His/H,-adenine/Ade）;100,10-1,and 10-2 indicates concentration of E.coli;pGBKT7-53 and pGADT7-T are used as positive control;pGBKT7-Lam and pGADT7-T are used as negative control;4330 is abbreviated from Soltu.DM.11G004330）. B：LUC complementation of Soltu.DM.11G004330 and SLF.（The left figure denotes how injections were carried out on tobacco leaves and 4330 was the abbreviation of Soltu.DM.11G004330）

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