基于转录组的桑树GRAS基因家族鉴定及MnGRAS22盐胁迫响应分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-1191

摘要/Abstract

摘要：

目的鉴定桑树MnGRAS基因家族成员，解析其盐胁迫响应表达规律与功能特征，为桑树耐盐遗传改良提供理论支撑。方法基于桑树盐胁迫转录组数据筛选MnGRAS基因；通过生物信息学分析其理化性质、系统发育分类、基因结构特征及启动子顺式作用元件；结合转录组与RT-qPCR验证基因表达模式；构建MnGRAS22过表达/抑制表达体系，通过生理生化指标测定明确其耐盐功能。结果成功鉴定出40个MnGRAS基因，归为10个亚家族，所有成员均含典型GRAS结构域；启动子区富集生长发育及非生物胁迫响应相关顺式作用元件。盐胁迫条件下，92.5%的基因可检测到表达信号，仅少数基因（MnGRAS11/27/34）在各胁迫时间点均无表达。9个候选基因中，66.7%的基因在叶片中持续上调表达，在根和茎中多呈动态表达特征；其中MnGRAS22在叶片和根系中的表达量分别为对照的88倍和36倍。过表达MnGRAS22可激活氧化酶基因（SOD、POD）表达，显著提高植株超氧化物歧化酶（superoxide dismutase, SOD）、过氧化物酶（peroxidase, POD）、过氧化氢酶（catalase, CAT）活性，促进脯氨酸（proline, Pro）积累，降低丙二醛（malondialdehyde, MDA）、超氧阴离子（superoxide anion radical, O₂^•-）和过氧化氢（hydrogen peroxide, H₂O₂）含量。结论桑树中鉴定出40个MnGRAS基因，MnGRAS22在盐胁迫下于叶片和根系中高表达；该基因过表达可通过增强抗氧化能力、调节渗透平衡、减轻膜损伤，显著提升植株耐盐性。

关键词: 桑树, GRAS基因家族, MnGRAS22, 转录组, 盐胁迫, 基因鉴定, 抗氧化响应, 耐盐性

Abstract:

Objective This study aimed to identify members of the MnGRAS gene family in mulberry (Morus alba), clarify their expression patterns and functional characteristics in response to salt stress, and provide theoretical support for the genetic improvement of salt tolerance in mulberry. Method MnGRAS genes were screened from mulberry transcriptome data under salt stress. Bioinformatic analyses were conducted to characterize their physicochemical properties, phylogenetic classification, gene structural features, and promoter cis-acting elements. Gene expression patterns were verified by integrating transcriptome data with RT-qPCR. The overexpression and RNA interference (RNAi)-mediated suppression systems of MnGRAS22 were constructed, and its salt-tolerance function was elucidated by determining physiological and biochemical indices. Result A total of 40 MnGRAS genes were successfully identified and classified into 10 subfamilies, with all members containing the typical GRAS domain. Promoter regions were enriched with cis-acting elements associated with growth, development, and abiotic stress responses. Under salt stress, 92.5% genes showed detectable expression signals, while only a few genes (MnGRAS11, MnGRAS27, MnGRAS34) had no expression at any stress time point. Among the 9 candidate genes, 66.7% were continuously upregulated in the leaves, whereas most showed dynamic expression patterns in the roots and stems. Specifically, the expressions of MnGRAS22 in the leaves and roots were 88-fold and 36-fold higher than those in the control, respectively. The overexpression of MnGRAS22 activated the expressions of oxidase genes (SOD, POD), significantly increased the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in plants, promoted proline accumulation, and reduced the contents of malondialdehyde (MDA), superoxide anion radical (O₂^•-), and hydrogen peroxide (H₂O₂). Conclusion Forty MnGRAS genes were identified in mulberry, among which MnGRAS22 was highly expressed in the leaves and roots under salt stress. The overexpression of MnGRAS22 can significantly enhance plant salt tolerance by improving antioxidant capacity, regulating osmotic balance, and alleviating membrane damage.

Key words: mulberry, GRAS gene family, MnGRAS22, transcriptome, salt stress, gene identification, antioxidant response, salt tolerance

董亚茹, 修妤, 赵东晓, 朱红, 朱琳, 刘惠芬. 基于转录组的桑树GRAS基因家族鉴定及MnGRAS22盐胁迫响应分析[J]. 生物技术通报, 2026, 42(7): 193-203.

DONG Ya-ru, XIU Yu, ZHAO Dong-xiao, ZHU Hong, ZHU Lin, LIU Hui-fen. Transcriptome-wide Identification of the GRAS Gene Family in Mulberry and Analysis of MnGRAS22 in Response to Salt Stress[J]. Biotechnology Bulletin, 2026, 42(7): 193-203.

图/表 8

图1 桑树和拟南芥GRAS家族成员的系统进化分析红色五角星和黑色三角分别表示桑树和拟南芥GRAS

Fig. 1 Phylogenetic analysis of GRAS family members from mulberry and ArabidopsisThe red stars indicates the GRAS of mulberry and black triangle indicates Arabidopsis, respectively

图2 桑树MnGRAS基因家族保守基序（A）、结构域（B）及基因结构（C）分析

Fig. 2 Conserved motif (A), domains (B), and gene structure (C) analysis of MnGRAS gene family in mulberry

图3 桑树GRAS基因启动子顺式作用元件分布

Fig. 3 Distribution of cis-acting elements of GRAS gene promoter in mulberry

图4 桑树GRAS响应盐胁迫的表达模式热图为200 mmol/L NaCl处理下桑树叶片中GRAS基因的表达水平。红色代表高表达，蓝色代表低表达

Fig. 4 Expression patterns of GRAS in mulberry in response to salt stressHeatmap showing expressions of GRAS genes in mulberry leaves under 200 mmol/L NaCl treatment. Red indicates high expression, and blue indicates low expression

图5 盐胁迫下桑树GRAS基因组织特异性表达分析*表示组内处理组与对照组在P<0.05水平上差异显著。下同

Fig. 5 Tissue-specific expression analysis of GRAS genes in mulberry under salt stress* indicates significant difference at the P<0.05 level between the treatment group and the control group within the same group. The same below

图6 MnGRAS22瞬时转化桑树植株的RT-qPCR检测

Fig. 6 RT-qPCR detection of mulberry plants transiently transformed with MnGRAS22

图7 MnGRAS22基因耐盐性分析A：SOD活性；B：POD活性；C：CAT活性；D：MDA含量；E：脯氨酸含量；F：DAB组织化学染色；G：NBT组织化学染色；H：Evans Blue组织化学染色

Fig. 7 Salt-tolerance analysis of MnGRAS22 geneA: SOD activity; B: POD activity; C: CAT activity; D: MDA content; E: Proline content; F: DAB histochemical staining; G: NBT histochemical staining; H: Evans blue histochemical staining

图8 抗氧化酶基因表达分析

Fig. 8 Expression analysis of antioxidant enzyme genes

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