技术领域Technical Field
本发明涉及植物基因工程技术领域，具体涉及紫花苜蓿MsGOLS2基因及其应用。The invention relates to the technical field of plant genetic engineering, and in particular to an alfalfa MsGOLS2 gene and an application thereof.
背景技术Background Art
紫花苜蓿(Medicago sativa L.)是种植最广泛的多年生牧草豆科植物，主要分布于北部、西北和东北地区，其根系发达，耐盐，抗逆性强，是有极高营养价值的牧草。此外，苜蓿作为生产乙醇的生物燃料原料，在未来能源革命中具有巨大潜力。然而，由于土地盐碱化程度的增加，多种野生苜蓿的生产力和生长持久性受到了极大影响。因此，深入研究苜蓿适应盐胁迫的分子机制，发掘抗逆基因对改良当地苜蓿育种结构、提高盐渍化土地的利用率至关重要。Alfalfa (Medicago sativa L.) is the most widely planted perennial forage legume, mainly distributed in the north, northwest and northeast regions. It has a well-developed root system, is salt-tolerant and has strong stress resistance, and is a forage with extremely high nutritional value. In addition, alfalfa, as a biofuel raw material for the production of ethanol, has great potential in the future energy revolution. However, due to the increasing degree of land salinization, the productivity and growth persistence of many wild alfalfa species have been greatly affected. Therefore, in-depth research on the molecular mechanism of alfalfa's adaptation to salt stress and the discovery of stress-resistant genes are crucial to improving the local alfalfa breeding structure and increasing the utilization rate of salinized land.
GOLS以基因家族形式存在于植物中，现已在拟南(Arabidopsis thaliana)、玉米(Zea mays)、牛耳草(Boea hygrometrica)、豌豆(Pisum sativum)、酿酒葡萄(Vitisvinifera)、黄花苜蓿(Medicago falcate)等物种中鉴定到不同的GOLS基因。在拟南芥中，存在7个编码GOLS的基因，它们在响应不同非生物胁迫时表现出特异性。其中，AtGOLS1受高温、高盐和干旱诱导，AtGOLS2仅受干旱和高盐胁迫诱导，而AtGOLS3仅在低温胁迫条件下表达。在转基因拟南芥植物中，由于AtGOLS2过表达引起GOLS活性和RFOs水平增高，表现为植株叶片脱水耐性增加。Salvi等人研究发现，在鹰嘴豆中，GOLS酶活性以及随之生成的半乳糖醇和棉子糖含量在遭受非生物胁迫后显著升高，并通过抑制植物中过量ROS积累和脂质过氧化反应来缓解胁迫造成的生长抑制。Santos等人报道，咖啡中GOLS基因家族成员(CaGolS1，CaGolS2，CaGolS3)通过提高棉子糖和水苏糖含量来响应干旱、高盐和高温等非生物胁迫。玉米基因组有三个编码肌醇半乳糖苷合成酶的基因，其中ZmGOLS3在种子成熟过程中表达，其转录物在成熟种子中积累。Zhuo等人研究表明，黄花苜蓿MfGOLS1在植物响应低温胁迫起重要作用。迄今为止，虽然在拟南芥、玉米等植物中GOLS基因已经被分离，对其功能的研究较为深入，但目前对该基因表达调控机制的研究鲜有报道，尤其是在紫花苜蓿中对GOLS响应盐胁迫的分子机制仍不清楚。因此对紫花苜蓿GOLS基因上游调控因子的鉴定及在植物抗逆中的作用机理需要进行深入的研究。GOLS exists in plants as a gene family. Different GOLS genes have been identified in species such as Arabidopsis thaliana, Zea mays, Boea hygrometrica, Pisum sativum, Vitis vinifera, and Medicago falcate. In Arabidopsis, there are seven genes encoding GOLS, which show specificity in response to different abiotic stresses. Among them, AtGOLS1 is induced by high temperature, high salt, and drought, AtGOLS2 is only induced by drought and high salt stress, and AtGOLS3 is only expressed under low temperature stress. In transgenic Arabidopsis plants, the overexpression of AtGOLS2 causes an increase in GOLS activity and RFOs levels, which is manifested as increased dehydration tolerance of plant leaves. Salvi et al. found that in chickpea, GOLS enzyme activity and the resulting galactitol and raffinose content increased significantly after abiotic stress, and alleviated the growth inhibition caused by stress by inhibiting excessive ROS accumulation and lipid peroxidation in plants. Santos et al. reported that members of the GOLS gene family (CaGolS1, CaGolS2, CaGolS3) in coffee responded to abiotic stresses such as drought, high salt and high temperature by increasing the content of raffinose and stachyose. The corn genome has three genes encoding inositol galactosidase synthase, of which ZmGOLS3 is expressed during seed maturation and its transcripts accumulate in mature seeds. Zhuo et al. showed that MfGOLS1 of yellow alfalfa plays an important role in plant response to low temperature stress. So far, although GOLS genes have been isolated in plants such as Arabidopsis and corn, and their functions have been studied in depth, there are few reports on the regulation mechanism of gene expression, especially the molecular mechanism of GOLS response to salt stress in alfalfa is still unclear. Therefore, in-depth research is needed to identify the upstream regulatory factors of the alfalfa GOLS gene and its mechanism of action in plant stress resistance.
发明内容Summary of the invention
针对现有技术中的上述不足，本发明的目的在于提供紫花苜蓿MsGOLS2基因及其在调控植物耐盐性中应用。In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an alfalfa MsGOLS2 gene and its application in regulating plant salt tolerance.
为了达到上述发明目的，本发明采用的技术方案为：In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:
第一方面，提供紫花苜蓿MsGOLS2基因，其核苷酸序列如SEQ ID NO.1所示。In a first aspect, a gene of MsGOLS2 from alfalfa is provided, and the nucleotide sequence thereof is shown as SEQ ID NO.1.
第二方面，提供含有紫花苜蓿MsGOLS2基因的转基因载体。In a second aspect, a transgenic vector containing the alfalfa MsGOLS2 gene is provided.
第三方面，提供含有紫花苜蓿MsGOLS2基因的重组菌株或重组细胞。In a third aspect, a recombinant strain or recombinant cell containing the alfalfa MsGOLS2 gene is provided.
第四方面，提供紫花苜蓿MsGOLS2基因过表达在提高植物耐盐性中的应用。In a fourth aspect, the invention provides the application of overexpression of MsGOLS2 gene of alfalfa in improving the salt tolerance of plants.
第五方面，提供紫花苜蓿MsGOLS2基因在紫花苜蓿抗性品质改良及耐盐紫花苜蓿优良品种选育中的应用。The fifth aspect provides the application of the alfalfa MsGOLS2 gene in improving the resistance quality of alfalfa and breeding of salt-tolerant alfalfa varieties.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明从紫花苜蓿中克隆出了与耐盐性相关的MsGOLS2基因，构建了MsGOLS2基因的重组载体，并在拟南芥中验证了MsGOLS2基因提高植物耐盐性功能，本发明可用于改良苜蓿的耐盐性，创制耐盐性强的紫花苜蓿种质材料。The invention cloned the MsGOLS2 gene related to salt tolerance from alfalfa, constructed a recombinant vector of the MsGOLS2 gene, and verified the function of the MsGOLS2 gene in improving plant salt tolerance in Arabidopsis thaliana. The invention can be used to improve the salt tolerance of alfalfa and create alfalfa germplasm material with strong salt tolerance.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为实施例中0h下MsGOLS2基因在不同组织中的表达量；S:茎；L：叶；R：根；Figure 1 shows the expression levels of MsGOLS2 gene in different tissues at 0h in the embodiment; S: stem; L: leaf; R: root;
图2为实施例中200mmol·L-1NaCl胁迫不同时间MsGOLS2基因在紫花苜蓿组织的表达量；FIG2 shows the expression level of MsGOLS2 gene in alfalfa tissue under 200 mmol·L-1 NaCl stress at different times in the embodiment;
图3为实施例中转基因拟南芥半定量RT-PCR，L2、L4、L6、L7、L8为不同的转基因株系，WT为野生型；FIG3 is a semi-quantitative RT-PCR of transgenic Arabidopsis thaliana in the embodiment, L2, L4, L6, L7, L8 are different transgenic strains, and WT is the wild type;
图4为实施例中75mM NaCl胁迫下野生型拟南芥(WT)和转基因拟南芥表型图，L4、L6、L7为不同的转基因株系；FIG4 is a phenotype diagram of wild-type Arabidopsis thaliana (WT) and transgenic Arabidopsis thaliana under 75 mM NaCl stress in the embodiment, where L4, L6, and L7 are different transgenic strains;
图5为实施例中野生型拟南芥(WT)和转基因拟南芥(GOLS2)根长统计分析，L4、L6、L7为不同的转基因株系。FIG5 is a statistical analysis of root length of wild-type Arabidopsis thaliana (WT) and transgenic Arabidopsis thaliana (GOLS2) in the example, where L4, L6, and L7 are different transgenic lines.
具体实施方式DETAILED DESCRIPTION
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.
实施例Example
1、紫花苜蓿MsGOLS2基因的克隆及序列鉴定1. Cloning and sequence identification of MsGOLS2 gene from alfalfa
紫花苜蓿盐胁迫前后转录组测序分析，筛选盐胁迫关键响应基因GOLS2。具体地说，转录组测序材料为紫花苜蓿根组织，经过200mM NaCl盐处理0h、2h、4h、8h后，采用本实验室改良过的Trizol法，分别提取各样本的总RNA，使用Nanodrop 2000超微量分光光度计和Agilent 2100生物分析仪对RNA进行定量及质量检测；以上每组样品包括3个生物学重复，共12个样本；采用Illumina Hiseq X Ten平台进行双端测序，通过HISAT2软件比对参考基因组，根据比对结果，利用Stringtie重构转录本，并利用RSEM计算每个样本中所有基因的表达量，以FDR<0.05且|log2FC|>1作为筛选条件，使用DESeq2软件分析差异表达基因。转录组数据分析发现肌醇半乳糖苷合酶GOLS2在盐胁迫下表达量显著升高。Transcriptome sequencing analysis of alfalfa before and after salt stress was performed to screen the key salt stress response gene GOLS2. Specifically, the transcriptome sequencing material was alfalfa root tissue. After 200mM NaCl salt treatment for 0h, 2h, 4h, and 8h, the total RNA of each sample was extracted using the Trizol method modified by our laboratory, and the RNA was quantified and quality tested using the Nanodrop 2000 ultra-micro spectrophotometer and the Agilent 2100 bioanalyzer; each group of samples above included 3 biological replicates, for a total of 12 samples; Illumina Hiseq X Ten platform was used for paired-end sequencing, and the reference genome was aligned using the HISAT2 software. According to the alignment results, the transcripts were reconstructed using Stringtie, and the expression levels of all genes in each sample were calculated using RSEM. FDR<0.05 and |log2FC|>1 were used as screening conditions, and the differentially expressed genes were analyzed using the DESeq2 software. Transcriptome data analysis found that the expression level of inositol galactosidase GOLS2 was significantly increased under salt stress.
利用SnapGene 6.0.2软件设计GOLS2基因全长扩增引物GOLS2-F/R。根据RNA提取试剂盒的操作说明提取紫花苜蓿总RNA，并反转出cDNA。以cDNA为模板进行MsGOLS2基因的CDS序列克隆，反应体系：50μL，cDNA模板2μL，MsGOLS2引物各1μL，2×Phanta Flash MasterMix(Dye Plus)25μL，双蒸水21μL：反应程序：98℃30s，98℃10s，55℃5s，72℃10s(32个循环)，72℃1min。将克隆产物进行电泳检测后，连接到pTOPO载体上，转化到大肠杆菌DH5α内，在含有氨苄霉素的固体LB上过夜生长，挑取单菌落进行菌落PCR鉴定，同时进行摇菌，将PCR结果为阳性的对应菌液送至北京华大基因公司进行测序。利用SnapGene 6.0.2软件将测序得到的序列进行拼接，获得MsGOLS2基因的正确序列(SEQ ID NO.1)，并对其进行翻译得到氨基酸序列。The full-length amplification primers GOLS2-F/R of GOLS2 gene were designed by SnapGene 6.0.2 software. Total RNA of alfalfa was extracted according to the operating instructions of RNA extraction kit, and cDNA was reversed. CDS sequence of MsGOLS2 gene was cloned with cDNA as template, reaction system: 50μL, cDNA template 2μL, MsGOLS2 primers 1μL each, 2×Phanta Flash MasterMix (Dye Plus) 25μL, double distilled water 21μL: reaction program: 98℃30s, 98℃10s, 55℃5s, 72℃10s (32 cycles), 72℃1min. After electrophoresis detection, the cloned product was connected to pTOPO vector, transformed into Escherichia coli DH5α, grown overnight on solid LB containing ampicillin, single colonies were picked for colony PCR identification, and the bacteria were shaken at the same time. The corresponding bacterial solution with positive PCR results was sent to Beijing BGI for sequencing. The sequences obtained by sequencing were spliced using SnapGene 6.0.2 software to obtain the correct sequence of the MsGOLS2 gene (SEQ ID NO.1), which was then translated to obtain the amino acid sequence.
2、MsGOLS2基因在紫花苜蓿中的表达模式分析2. Analysis of the expression pattern of MsGOLS2 gene in alfalfa
将紫花苜蓿“中苜1号”种子在土壤中播种10d后，转移至含有霍格兰营养液的水培盒中继续生长12d。选择生长状况良好、大小一致的植株进行200mmol·L-1NaCl处理，分别处理0、2、4、8个小时，最后统一收样，液氮速冻后放置-80℃冰箱内保存。根据RNA提取试剂盒的操作说明提取紫花苜蓿总RNA，并反转出cDNA，方法同1。利用SnapGene 6.0.2软件设计GOLS2基因荧光定量PCR引物GOLS2-qPCR-F1/R1，以紫花苜蓿MsActin-F/R为内参基因引物，进行荧光定量PCR(qRT-PCR)。反应体系20μL，2×ChamQ Universal SYBR PCR Master Mix10μL，荧光定量PCR引物各0.4μL，cDNA2μL，双蒸水7.2μL。反应程序：第一步预变性：95℃30s；第二步循环反应：95℃10s、60℃30s，循环40次；第三步溶解曲线：95℃15s、60℃60s、95℃15s。将得到的Ct值用2-ΔΔCT方法计算MsGOLS2基因在不同组织的表达量以及NaCl处理后不同时间段根中MsGOLS2基因的表达量，其中ΔΔCT＝(CTNaCl处理的目的基因-CTNaCl处理的内参基因)-(CT对照组的目的基因-CT对照组的内参基因)。After 10 days of sowing in the soil, the seeds of alfalfa "Zhongmu No. 1" were transferred to a hydroponic box containing Hoagland's nutrient solution and continued to grow for 12 days. Plants with good growth and uniform size were selected for 200mmol·L-1 NaCl treatment for 0, 2, 4, and 8 hours, and finally collected uniformly, frozen in liquid nitrogen, and stored in a -80℃ refrigerator. According to the operating instructions of the RNA extraction kit, total RNA of alfalfa was extracted and cDNA was reversed, with the same method as 1. SnapGene 6.0.2 software was used to design the GOLS2 gene fluorescence quantitative PCR primer GOLS2-qPCR-F1/R1, and alfalfa MsActin-F/R was used as the internal reference gene primer for fluorescence quantitative PCR (qRT-PCR). The reaction system was 20μL, 2×ChamQ Universal SYBR PCR Master Mix 10μL, fluorescence quantitative PCR primers 0.4μL each, cDNA 2μL, and double distilled water 7.2μL. Reaction procedure: first step pre-denaturation: 95℃30s; second step cycle reaction: 95℃10s, 60℃30s, 40 cycles; third step melting curve: 95℃15s, 60℃60s, 95℃15s. The obtained Ct value was used to calculate the expression of MsGOLS2 gene in different tissues and the expression of MsGOLS2 gene in roots at different time periods after NaCl treatment using the 2-ΔΔCT method, where ΔΔCT = (target gene treated with CTNaCl-reference gene treated with CT NaCl)-(target gene in the CT control group -reference gene in the CT control group ).
3、紫花苜蓿MsGOLS2基因转基因载体构建3. Construction of transgenic vector of MsGOLS2 gene in alfalfa
以1中获得的含有MsGOLS2基因正确序列的质粒为模板，以含有载体UBQ10双酶切位点同源臂的UBQ10-GOLS2-HR-F/R为引物，用诺唯赞公司高保真酶P520克隆MsGOLS2基因全长。用限制性内切酶Nco I和Spe I双酶切质粒UBQ10，用诺威赞的同源重组试剂盒将带有同源臂的MsGOLS2基因片段和双酶切好的UBQ10载体进行连接，并转化至大肠杆菌DH5α中，测序正确后提取质粒，即为UBQ10-GOLS2重组质粒。The plasmid containing the correct sequence of the MsGOLS2 gene obtained in 1 was used as a template, and UBQ10-GOLS2-HR-F/R containing the homology arms of the double restriction site of the vector UBQ10 was used as a primer, and the high-fidelity enzyme P520 of Novazon was used to clone the full length of the MsGOLS2 gene. The plasmid UBQ10 was double-digested with restriction endonucleases Nco I and Spe I, and the MsGOLS2 gene fragment with homology arms and the double-digested UBQ10 vector were connected using the homologous recombination kit of Novazon, and transformed into Escherichia coli DH5α. After the sequencing was correct, the plasmid was extracted, which was the UBQ10-GOLS2 recombinant plasmid.
4、转基因拟南芥的获得、筛选、鉴定和耐盐性分析4. Acquisition, screening, identification and salt tolerance analysis of transgenic Arabidopsis
将重组质粒转化到农杆菌GV3101中，通过花侵染将MsGOLS2基因转入野生型拟南芥。收获后的T0代拟南芥种子在含有潮霉素(Hygromycin B，40mg·L-1)的1/2MS固体培养基上逐代筛选，最终获得能够稳定表达的T2代纯合种子用于后续试验。培养纯合植株提取RNA并反转录获得cDNA，以MsGOLS2-SQ-F/R为特异性引物，以AtActinF/R为内参引物，用半定量RT-PCR技术检测转基因拟南芥中MsGOLS2基因的表达情况。The recombinant plasmid was transformed into Agrobacterium GV3101, and the MsGOLS2 gene was transferred into wild-type Arabidopsis by floral infection. The harvestedT0 generation Arabidopsis seeds were screened generation by generation on 1/2MS solid medium containing hygromycin B (40 mg·L-1 ), and finally theT2 generation homozygous seeds with stable expression were obtained for subsequent experiments. Homozygous plants were cultured to extract RNA and reverse transcribed to obtain cDNA. The expression of the MsGOLS2 gene in transgenic Arabidopsis was detected by semi-quantitative RT-PCR using MsGOLS2-SQ-F/R as specific primers and AtActinF/R as internal reference primers.
将野生型拟南芥种子WT和获得的T2代转基因种子用75％的酒精在超净工作台中消毒20min，然后用无菌水清洗5遍，均匀的点播在1/2MS固体培养基(MS粉2.23g·L-1，蔗糖15g，琼脂6.5g·L-1，pH＝6)上，在4℃冰箱中春化48h，然后转移至正常条件下竖直生长4d，选取生长状态一致的幼苗转移到含有0和75mmol·L-1NaCl的1/2MS固体培养基上继续生长，在转移过程中需要将野生型和转基因型幼苗转移到同一处理的培养基上，避免不同培养基之间的差异，3～4d后拍照记录并统计表型差异。利用ImageJ软件测量拟南芥幼苗的根长，GraphPad.Prism.9.5对数据进行统计分析。The wild-type Arabidopsis seeds WT and the obtained T2 transgenic seeds were sterilized with 75% alcohol in a clean bench for 20 minutes, then washed with sterile water for 5 times, and evenly sown on 1/2MS solid medium (MS powder 2.23g·L-1 , sucrose 15g, agar 6.5g·L-1 , pH=6), vernalized in a 4℃ refrigerator for 48h, and then transferred to normal conditions for vertical growth for 4d. The seedlings with the same growth status were selected and transferred to 1/2MS solid medium containing 0 and 75mmol·L-1 NaCl for further growth. During the transfer process, the wild-type and transgenic seedlings needed to be transferred to the same treated medium to avoid differences between different mediums. After 3-4 days, photos were taken to record and statistically analyze the phenotypic differences. The root length of Arabidopsis seedlings was measured using ImageJ software, and the data were statistically analyzed using GraphPad.Prism.9.5.
表1试验中用到的引物Table 1 Primers used in the experiment
5、实验结果5. Experimental results
以紫花苜蓿的根、茎、叶组织的cDNA为模板，利用qRT-PCR技术分析MsGOLS2在正常条件和200mmol·L-1NaCl胁迫条件下的相对表达量，正常条件下以表达量最低的茎为单位对照进行统计；200mmol·L-1NaCl胁迫条件下以表达量最低的0小时为对照进行统计。结果显示，正常条件下，MsGOLS2基因在根中的表达量显著高于茎，约48.3倍(图1)；盐胁迫8小时后，MsGOLS2基因在根中的表达量显著升高79.5倍(图2)，证明了MsGOLS2基因的组织表达特异性以及对盐胁迫的积极响应。Using cDNA of root, stem and leaf tissues of alfalfa as template, qRT-PCR technology was used to analyze the relative expression of MsGOLS2 under normal conditions and 200mmol·L-1 NaCl stress conditions. Under normal conditions, the stem with the lowest expression was used as the control for statistics; under 200mmol·L-1 NaCl stress conditions, the 0 hour with the lowest expression was used as the control for statistics. The results showed that under normal conditions, the expression level of MsGOLS2 gene in the root was significantly higher than that in the stem, about 48.3 times (Figure 1); after 8 hours of salt stress, the expression level of MsGOLS2 gene in the root increased significantly by 79.5 times (Figure 2), proving the tissue expression specificity of MsGOLS2 gene and its positive response to salt stress.
利用花侵染法将带有MsGOLS2基因的农杆菌(GV3101)转入野生型拟南芥(WT)中，在含有潮霉素(Hygromycin B，50mg·L-1)的1/2MS培养基上逐代筛选出能够稳定表达的T2代纯合种子，共筛选获得21个纯合株系。随机挑选其中5个株系进行半定量RT-PCR检测转基因拟南芥中MsGOLS2基因的表达情况，其结果如图3所示。所挑选的5个转基因株系中都有目的基因的条带，而野生型拟南芥没有MsGOLS2基因的条带，表明本试验成功的将该基因在拟南芥中表达。Agrobacterium (GV3101) carrying the MsGOLS2 gene was transferred into wild-type Arabidopsis thaliana (WT) by floral infection, and the homozygous seeds of theT2 generation with stable expression were screened generation by generation on 1/2MS medium containing hygromycin B (50 mg·L-1 ), and a total of 21 homozygous strains were screened. Five of the strains were randomly selected for semi-quantitative RT-PCR to detect the expression of the MsGOLS2 gene in transgenic Arabidopsis thaliana, and the results are shown in Figure 3. The five selected transgenic strains all had bands of the target gene, while the wild-type Arabidopsis did not have bands of the MsGOLS2 gene, indicating that this experiment successfully expressed the gene in Arabidopsis thaliana.
为了进一步验证MsGOLS2基因对拟南芥幼苗耐盐性的影响，本试验将在1/2MS培养基上生长了4d的幼苗转移到含有0和75mmol·L-1NaCl的1/2MS固体培养基上，继续生长一段时间，每天观察各个株系的表型。结果如图4-5所示，在正常的1/2MS固体培养基上野生型与转基因型拟南芥幼苗没有明显差别，在含有75mmol·L-1NaCl的1/2MS固体培养基上，转基因型拟南芥幼苗的根显著长于WT(P<0.01)，表明在盐胁迫下诱导了MsGOLS2基因的表达，证明了MsGOLS2基因的过表达提高了拟南芥的耐盐性。In order to further verify the effect of MsGOLS2 gene on the salt tolerance of Arabidopsis seedlings, this experiment transferred the seedlings grown on 1/2MS medium for 4 days to 1/2MS solid medium containing 0 and 75mmol·L-1 NaCl, and continued to grow for a period of time, and observed the phenotype of each strain every day. The results are shown in Figures 4-5. There is no significant difference between wild-type and transgenic Arabidopsis seedlings on normal 1/2MS solid medium. On 1/2MS solid medium containing 75mmol·L-1 NaCl, the roots of transgenic Arabidopsis seedlings are significantly longer than WT (P<0.01), indicating that the expression of MsGOLS2 gene is induced under salt stress, proving that overexpression of MsGOLS2 gene improves the salt tolerance of Arabidopsis.
于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.
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| CN202411105027.7ACN118726419A (en) | 2024-08-13 | 2024-08-13 | MsGOLS2 gene from alfalfa and its application in regulating plant salt tolerance |
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