ZmSTART1调控玉米维管束建成的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0981

摘要/Abstract

摘要：

目的解析玉米基因ZmSTART1的结构，明确ZmSTART1的表达特性，分析ZmSTART1在维管束建成过程中的作用，为玉米抗倒伏和产量性状的遗传改良提供理论基础。方法利用生物信息学方法，解析ZmSTART1的结构特征；利用Real-Time PCR技术分析ZmSTART1的时空表达特性；利用ZmSTART1-GFP融合技术转化烟草叶片，对ZmSTART1进行亚细胞定位；构建ZmSTART1过表达载体，利用农杆菌侵染花絮法转化Col-0拟南芥，利用Real-Time PCR技术验证过表达ZmSTART1拟南芥阳性苗，观察过表达ZmSTART1拟南芥的维管束特征，明确ZmSTART1调控维管束建成的生物学功能。结果生物信息学分析发现，ZmSTART1是一个仅含有1个START结构域的疏水性蛋白质。Real-Time PCR技术分析ZmSTART1的时空表达特性发现，ZmSTART1在花药、叶片和第一节间中表达量较高。亚细胞定位表明，ZmSTART1定位于细胞质膜上。创制过表达ZmSTART1的拟南芥植株ZmSTART1-OE发现，过表达ZmSTART1-OE转基因拟南芥子叶叶脉和真叶二、三级叶脉个数均比野生型拟南芥多，而且叶脉形成的封闭空间数也显著高于野生型对照，ZmSTART1-OE转基因拟南芥叶片出现四级叶脉时，野生型对照叶片只有三级叶脉。观察茎维管束发育情况发现，野生型拟南芥茎初生结构内维管束为6个，ZmSTART1-OE茎初生结构维管束增多为7个，束间纤维细胞层数减少、番红染色变浅、木质素含量降低。结论 ZmSTART1属于START家族成员，定位于细胞质膜，在玉米的维管束建成过程中具有重要作用。

关键词: ZmSTART1, 表达特性, 维管束建成, 玉米, 拟南芥

Abstract:

Objective The objective of this study is to analyze the structure of ZmSTART1, clarify the expressing characteristics of ZmSTART1, analyze the function of ZmSTART1 in the process of vascular bundle construction, and thus provide a theoretical basis for the genetic improvement of corn lodging resistance and yield traits. Method Bioinformatics method was applied to analyze the structure characteristics of ZmSTART1 protein. Real-time PCR was used to analyze the temporal and spatial expression of ZmSTART1. ZmSTART1-GFP fusion technology was to transform tobacco leaves and determine the subcellular location of ZmSTART1. The overexpressing vector of ZmSTART11 was constructed, and Agrobacterium infection flocculent method was to transform Col-0 Arabidopsis thaliana. Real-time PCR technology was applied to validate the ZmSTART1 overexpressed A.thaliana positive seedlings. The vascular bundle characteristics of A. thaliana overexpressing ZmSTART1 were observedand the biological function of ZmSTART1 in regulating vascular bundle formation was clarified. Result Bioinformatics analysis revealed that ZmSTART1 was a hydrophobic protein containing only one START domain. The spatiotemporal expression characteristics of ZmSTART1 via real-time PCR technology showed that ZmSTART1 had higher expressions in the anthers, leaves, and the first internode. Subcellular localization analysis revealed that ZmSTART1 was located on the cytoplasmic membrane. Creating A.thaliana plants ZmSTART1-OE overexpressing the ZmSTART1 gene, it was found that transgenic A.thaliana overexpressing ZmSTART1-OE had more secondary and tertiary leaf veins than wild-type ones, and the number of enclosed spaces formed by the leaf veins was also significantly higher than that of the wild-type control. When ZmSTART1-OE transgenic A.thaliana leaves showed fourth order leaf veins, the wild-type control leaves only had third order leaf veins. Observing the development of stem vascular bundles, it was found that the wild-type A. thaliana had 6 vascular bundles in its primary stem structure, while ZmSTART1-OE had 7 vascular bundles in its primary stem structure. The number of fiber cell layers between bundles decreased, the safranin staining became lighter, and the lignin content decreased. Conclusion ZmSTART1 belongs to the START family and is located on the cytoplasmic membrane, playing an important role in the process of vascular bundle formation in maize.

Key words: ZmSTART1, expression characteristics, vascular bundle formation, maize, Arabidopsis thaliana

刘彤彤, 李肖慧, 杨骏龙, 陈旺, 玉猛, 王超凡, 王凤茹, 客绍英. ZmSTART1调控玉米维管束建成的功能研究[J]. 生物技术通报, 2025, 41(4): 115-122.

LIU Tong-tong, LI Xiao-hui, YANG Jun-long, CHEN Wang, YU Meng, WANG Chao-fan, WANG Feng-ru, KE Shao-ying. Functional Study on ZmSTART1 Regulation of Maize Vascular Bundle Formation[J]. Biotechnology Bulletin, 2025, 41(4): 115-122.

图/表 10

表1 形态指标统计学分析

Table 1 Statistical analysis of morphological indexes

类型 Type	真叶面积 True leaf area/mm²	子叶面积 Cotyledon area/mm²	叶柄长 Petiole length/cm	株高 Plant height/cm	茎面积 Stem area/mm²	茎粗 Stem circumference/mm
WT	6.15±1.06aa	8.35±0.64aa	0.25±0.04aa	11±1.00aa	0.62±0.01aa	2.79±0.03aa
ZmSTART1-OE1	9.4±0.28AA	6.15±0.47AA	0.35±0.05AA	26.7±1.00AA	0.67±0.11aa	2.89±0.05aa
ZmSTART1-OE2	8.8±0.11AA	5.87±0.24AA	0.31±0.08Aa	22.5±1.87AA	0.63±0.13aa	2.81±0.17aa
ZmSTART1-OE3	8.8±0.23AA	5.15±0.33AA	0.28±0.07aa	18.6±1.65AA	0.62±0.07aa	2.79±0.13aa

图1 ZmSTART1结构分析A：ZmSTART1蛋白的结构域示意图；B：ZmSTART1基因的外显子和内含子示意图；C：ZmSTART1的三级结构

Fig. 1 Structure analysis of ZmSTART1A: Schematic diagram of the domain in ZmSTART1 protein. B: Schematic diagram of the exons and introns in ZmSTART1 gene. C: The tertiary structure of ZmSTART1

图2 拟南芥和玉米START家族成员系统发育进化树GRMZM：玉米；At：拟南芥

Fig. 2 Phylogenetic tree analysis of START genes of maize (Zea mays) and A. thalianaGRMZM: Zea mays; At: Arabidopsis thaliana

图3 玉米不同组织部位中ZmSTART1的表达V1：1片真叶展开期；V3：3片真叶展开期；V5：5片真叶展开期；V7：7片真叶展开期；R1：玉米的吐丝期。不同小写字母代表差异显著水平≤0.05。下同

Fig. 3 Expressions of ZmSTART1 in different tissues of maizeV1: 1 true leaf development stage. V3: 3 true leaf development stage. V5: 5 true leaf development stage. V7: 7 true leaf development stage. R1: Silking stage of maize. Different lowercase letters indicate a significant difference ≤0.05. The same below

图4 激光共聚焦显微镜下观察ZmSTART1-GFP的亚细胞分布

Fig. 4 The subcellular distribution of ZmSTART1-GFP observed under laser confocal microscopy (bar=50 μm)

图5 ZmSTART1阳性苗的筛选与鉴定A：潮霉素筛选；B：PCR鉴定；C：RT-qPCR表达量分析

Fig. 5 Screening and identification of ZmSTART1 positive seedlingsA: Hygromycin screening. B: PCR identification. C: Analysis of RT-qPCR expression

图6 过表达ZmSTART1转基因拟南芥的表型

Fig. 6 Phenotypic observation of transgenic linesoverexpressed ZmSTART1 in A. thaliana

表2 叶片叶脉、茎维管束表型数据统计

Table 2 Phenotype data statistics of superficial veins and vascular bundles

类型	维管束个数	子叶二级叶脉	真叶二级叶脉数	真叶三级叶脉数	真叶四级叶脉数	子叶脉间区域
Type	No. of vascular bundles	Cotyledon vein/Veins	Total number of secondary veins in true leaves	Total number of tertiary veins in true leaves	Total number of fourth-order veins of true leaves	Region between veins of cotyledons
WT	6aa	5±1aa	12±1aa	12±1aa	0aa	5±1aa
ZmSTART1-OE1	7±1Aa	7±1AA	17±2AA	37±7AA	4±1AA	7±1AA
ZmSTART1-OE2	7±1Aa	7±1AA	16±2AA	33±8AA	3±1AA	6±7Aa
ZmSTART1-OE3	6±1aa	6±1Aa	15±2Aa	30±8AA	2±1AA	5±7aa

图7 过表达ZmSTART1转基因拟南芥叶片叶脉表型A和B：分别为野生型（A）和过表达ZmSTART1转基因拟南芥（B）子叶和真叶叶脉；C和D：分别为野生型（C）和过表达ZmSTART1转基因拟南芥（D）真叶各级叶脉

Fig. 7 Phenotype of the of ZmSTART1-overexpressed transgenic A.thaliana leaf veinA and B: The cotyledon and true leaf veins of wild type (A) and ZmSTART1-overexpressed transgenic Arabidopsis (B) respectively. C and D: The true leaf veins at all levels of wild type (C) and ZmSTART1-overexpressed transgenic Arabidopsis (D) respectively

图8 过表达ZmSTART1转基因拟南芥茎横切面显微结构A和B：分别为野生型（A）和过表达ZmSTART1转基因拟南芥（B）茎横切面显微结构；C和D：分别为野生型（C）和过表达ZmSTART1转基因拟南芥（D）茎维管束

Fig. 8 Stem cross seetional microstructure of transgenie A. thaliana overexpressing ZmSTART1A and B: Stem cross seetional microstructure of wild type (A) and ZmSTART1-overexpressed transgenic Arabidopsis (B) respectively. C and D: The vascular bundle of wild type (C) and ZmSTART1-overexpressed transgenic Arabidopsis (D) respectively

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