紫花苜蓿非组培遗传转化体系创建及在耐盐基因功能鉴定与基因编辑中的应用

doi:10.11686/cyxb2025130

摘要/Abstract

摘要：

针对紫花苜蓿传统遗传转化技术存在的周期长、效率低、基因型依赖性强的技术瓶颈，本研究创新性地建立了基于发根农杆菌介导的高效非组培遗传转化体系。以‘龙牧806’苜蓿枝条为材料，采用优化的节下穿刺浸染法，构建无需组织培养的高效遗传转化体系。该方法可在14 d内获得转基因苜蓿嵌合体，生根率达72%~82%，毛状根遗传转化率达90.24%~94.59%。在应用验证方面，本研究取得两项重要突破：首先，利用该体系快速鉴定了耐盐基因MsRCI2D的功能，获得的转基因苜蓿嵌合体在200 mmol·L^-1NaCl胁迫下抗氧化酶活性显著提高、活性氧的积累显著下降、离子稳态调节能力增强；其次，结合可视化报告基因RUBY，建立了苜蓿基因编辑gRNA快速筛选技术，编辑效率达到23.07%。本研究建立的体系大幅缩短了转基因苜蓿嵌合体的获得周期，提高了转化效率。该技术不仅为紫花苜蓿基因功能研究提供了高效平台，同时为牧草分子设计育种提供了可靠的技术支撑，对其他饲草的遗传改良也具有重要参考价值。

关键词: 紫花苜蓿, 非组培转化, 转基因嵌合体, 耐盐基因, gRNA筛选

Abstract:

There are substantial challenges in the genetic improvement of alfalfa （Medicago sativa）， a globally important forage crop， because of the limitations of conventional transformation methods. These methods are time-consuming， genotype-dependent， and reliant on labor-intensive tissue culture processes. To address these issues， we developed a rapid， tissue culture-free transformation system for alfalfa. This system was developed and optimized using the alfalfa cultivar Longmu 806. The system employs Agrobacterium rhizogenes-mediated infection combined with a simple stem-pricking infiltration method. This innovative approach eliminates the need for callus induction and somatic embryogenesis， enabling the generation of transgenic chimeric alfalfa within just 14 days-a dramatic reduction compared with the 3-6 months typically required with traditional protocols. We used this system in two applications： 1） Rapid functional analysis of the MsRCI2D gene， which conferred enhanced salt tolerance in transgenic chimeric plants， as evidenced by increased antioxidant enzyme activities， decreased reactive oxygen species accumulation， and improved ion homeostasis under 200 mmol·L^-1NaCl stress； and 2） Establishment of an efficient guide RNA screening platform using the RUBY reporter system， which achieved a 23.07% editing efficiency in transformed roots， providing a robust and visual tool for optimizing CRISPR gRNAs. This breakthrough transformation strategy addresses major bottlenecks in alfalfa genetic engineering-namely genotype dependence and prolonged timelines—and offers a powerful platform for high-throughput gene function studies， abiotic stress tolerance improvement， and precision genome editing. By integrating simplicity， speed， and high efficiency， our system holds transformative potential for both fundamental research and molecular breeding in alfalfa and other recalcitrant forage crops.

Key words: Medicago sativa, tissue culture-free transformation, transgenic chimeras, salt tolerance, gRNA screening

张世超, 崔国文, 张德鹏, 韩福迎, 丁叮, 吕向丽, 林硕, 陈乐然, 李吉儒, 才华. 紫花苜蓿非组培遗传转化体系创建及在耐盐基因功能鉴定与基因编辑中的应用[J]. 草业学报, 2026, 35(3): 223-234.

Shi-chao ZHANG, Guo-wen CUI, De-peng ZHANG, Fu-ying HAN, Ding DING, Xiang-li LYU, Shuo LIN, Le-ran CHEN, Ji-ru LI, Hua CAI. Establishment of a tissue culture-free genetic transformation system for alfalfa and its applications in salt-tolerance gene functional characterization and gene editing[J]. Acta Prataculturae Sinica, 2026, 35(3): 223-234.

图/表 6

图1 MsPALM-1基因gRNA靶位点位置

Fig.1 The gRNA target sequence of MsPALM-1

图2 不同穿刺方式毛状根的生长情况（a）穿刺准备及过程Puncture preparation and procedure；（b）蘸菌与穿刺转化毛状根Dip and puncture transformed hairy roots；（c）节上与节下穿刺Subsegmental and segmental punctured hairy roots；（d）节下不同位置穿刺Different locations of below nodes punctured hairy roots；（e）节下0.5~1.0 cm处穿刺不同次数Different times of punctured hairy roots from 0.5-1.0 cm below the node.

Fig.2 Growth of hairy roots in different puncture methods

图3 不同转化方式处理14 d后毛状根生长情况（a）节上与节下穿刺Statistics on hairy root growth of subsegmental and segmental punctured branches；（b）不同位置节下穿刺Below nodes punctured branches at different locations；（c）节下0.5~1.0 cm处穿刺不同次数 Punctured branches with different number of times from 0.5-1.0 cm below the node. ****：差异极显著Extremely significant difference （P<0.0001）. 不同小写字母表示不同穿刺位置或不同穿刺次数间差异显著（P<0.05）。Different lowercase letters indicate significant differences （P<0.05） among different puncture locations or frequencies.

Fig.3 Statistical comparison of hairy root growth after 14 days of treatment by different transformation methods

图4 毛状根阳性苗鉴定（a）MsRCI2D基因的毛状根荧光Fluorescence observation of hairy roots of MsRCI2D gene；（b）毛状根阳性苗PCR鉴定PCR identification of hairy root positive seedlings；（c）阳性苗实时荧光定量PCR Real-time quantitative PCR of positive seedlings； M：分子量标记Trans 2K DNA Marker； -：阴性对照Negative control； +：阳性对照Positive control； WT：野生型植株Wild-type plant； D1~D3， D25， D26： MsRCI2D基因不同转化株系Plants transformed with MsSCI2D.**：差异极显著Extremely significant difference （P<0.01）.

Fig.4 Identification of hairy root positive seedlings

图5 盐胁迫下转MsRCI2D基因苜蓿嵌合体的表型分析D1， D2：不同转MsRCI2D植株Different trans-MsRCI2D plants； *：差异显著Significant difference （P<0.05）； **：差异极显著Extremely significant difference （P<0.01）.

Fig.5 Phenotypic analysis of alfalfa chimeras transgenic for the MsRCI2D gene under salt stress

图6 毛状根阳性苗鉴定及Sanger测序（a）红色毛状根表型Red hairy root phenotype；（b）毛状根阳性鉴定Hairy root positive identification；（c）Sanger测序结果Sanger sequencing results； M： DL2000 Marker； -：阴性对照Negative control； WT：野生型Wild type； +：阳性对照Positive control； 1~6：阳性苗鉴定Positive seedling identification； WT：野生型Wild type； MsPALM-1/2：阳性毛状根部分测序结果Positive trichome root sequencing results.

Fig.6 Identification of hairy root positive seedlings and sanger sequencing results

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