玉米ZCN7在调控花期抗旱性中的作用

Function of Maize ZCN7 in Regulating Drought Resistance at Flowering Stage

【目的】我国玉米主产区多分布在干旱和半干旱的雨养农业地区,干旱造成的玉米减产严重威胁国家粮食安全。作为异花授粉作物,开花期是玉米对干旱最为敏感的时期,干旱引起的散粉吐丝花期不遇将导致严重的产量损失。因此,挖掘花期抗旱基因,并研究其功能,将为玉米抗旱种质改良和品种培育提供理论支撑和基因资源。【方法】采取生物信息学方法,对玉米基因组中24个ZCN基因(拟南芥FT的同源基因)的同源关系进行分析。利用qRT-PCR和活体GFP荧光成像试验分析ZCN7的组织表达模式。选取118份玉米多样性自交系,在北京(2021和2022年)和新疆乌鲁木齐(2022年)3个环境下开展不同水分处理的玉米开花期性状调查,采用PCR扩增和Sanger测序方法对ZCN7及上下游区域的遗传变异进行检测,利用混合线性模型开展散粉-吐丝间隔的候选基因关联分析获得显著关联的遗传位点,同时,取花期叶片组织进行ZCN7表达量检测,分析显著位点不同单倍型自交系中散粉-吐丝间隔及ZCN7表达量的差别。构建Ubi1:ZCN7的过表达转基因玉米植株,在大田环境开展不同水分处理下的开花期鉴定和产量及相关性状的鉴定,分析ZCN7的花期抗旱功能。【结果】玉米24个ZCN基因被分为15个FT类基因、6个TFL1类基因和3个MFT类基因,编码的蛋白长度为111—193个氨基酸,其中,ZCN7与ZCN8的亲缘关系最近,二者蛋白序列的相似性为83.33%。组织表达分析结果表明,ZCN7在V12期表现出表达峰值,并且ZCN7的启动子在成熟的拟南芥叶片边缘高表达。候选基因关联分析发现位于ZCN7起始密码子前1001 bp的一个SNP位点具有最显著的关联信号,该位点AA和GG单倍型分别包含78和27个自交系,干旱条件下,A/A单倍型自交系的散粉-吐丝间隔显著小于G/G单倍型,并且A/A单倍型的玉米ZCN7表达量显著高于G/G单倍型。对ZCN7过表达转基因玉米的田间表型鉴定,表明干旱和正常水分条件下转基因玉米的散粉-吐丝间隔均小于野生型,其中,干旱条件下,OE1散粉-吐丝间隔较野生型缩小2.3 d,OE2较野生型缩小2.6 d;并且,在干旱条件下,转基因玉米的单株产量和单株籽粒数均显著高于野生型,而百粒重、粒长、粒宽等性状没有显著差异。【结论】玉米ZCN7正调控玉米的抗旱性,其过表达能够缩短干旱下的散粉-吐丝间隔,并增加籽粒产量。

【Objective】The main producing areas of maize is mostly located on the arid or semi-arid region that relying on the rainfed farming in China.The maize production losses caused by drought is a great threaten to food security.As a cross-pollinating crop,maize is mostly sensitive to water stress during flowering time.Drought at flowering stage will lead to asynchronous development between the male and female flower and cause massive grain yield loss.Thus,mining drought resistance related ge nes at flowering stage is important for maize drought resistance improvement and breeding.【Method】In the present study,the phylogenic tree of 24 ZCN genes in maize genome,which is homologs of Arabidopsis FT gene,was build.The gene expression patterns of ZCN7 were analysis using qRT-PCR and in vivo GFP fluorescence imaging.A maize natural population consisting of 118 diverse inbred lines were planted in three environments,Beijing in 2021 and 2022 and Urumqi in 2022,to identify the flowering time related traits under different water treatments.The genomic variants around ZCN7 were detected by PCR and Sanger sequencing.The candidate gene association analysis was performed based on mixed linear model and the significant associated variants with drought induced anthesis-silking interval was obtained.The gene expression level of ZCN7 in natural population at flowering time was also measured by qRT-PCR.The differences of drought resistance traits and ZCN7 expression were compared between different haplotypes of significant associated variant.The Ubi1:ZCN7 overexpression transgenic maize were obtained,and the phenotypic performance was identified under different water treatments.【Result】The 24 ZCN genes in maize genome included 15 FT like genes,6 TFL1 like genes and 3 MFT like genes.The protein sequence of ZCN genes varied from 111 nn to 193 nn.The ZCN7 showed close relationship with ZCN8 and the protein sequence identity was 83.3%between the two genes.ZCN7 showed highest gene expression in the leaf blade at V12 stage.And the ZCN7-promoter:GFP vector was transformed to Arabidopsis and the GFP showed enriched signal at the blade edge of mature leaf.The candidate gene association analysis revealed a SNP variant at 1001 bp upstream of ZCN7 start codon had highest association signal with drought induced anthesis-silking interval under drought.The A/A and G/G haplotypes of SNP-1001 included 78 and 27 inbred lines,respectively.The anthesis-silking interval of A/A haplotype lines were significantly lower than G/G lines.And the ZCN7 gene expression of A/A haplotype lines were significantly higher than G/G lines.In addition,the ZCN7 overexpression transgenic lines showed significantly decreased anthesis-silking interval than wild type lines.Under drought,the anthesis-silking intervals of OE1 and OE2were 2.3 and 2.6 days shorter than wild type lines.And the grain yield per plant and kernel number per plant of transgenic lines were significantly higher than wild type lines under drought,while the hundred kernel weight,kernel length and kernel width showed no significant difference.【Conclusion】The maize ZCN7 played positive role in drought resistance and its overexpression improved grain yield by reducing anthesis-silking interval under drought.