菠菜毛状根遗传转化体系建立及SoTPSs基因家族分析

【作者】 周敏；

【导师】 孙美红； 戴绍军；

【作者基本信息】 上海师范大学 ， 植物学， 2023， 硕士

【摘要】 菠菜（Spinacia oleracea L.）是耐寒不耐热的绿叶蔬菜,高温胁迫严重影响菠菜的生长、产量和品质。挖掘关键的菠菜耐热基因,将有助于利用分子育种策略进行菠菜耐热品种培育。然而,由于缺乏高效菠菜遗传转化体系,限制了菠菜耐热基因的功能鉴定和深入研究。我们通过筛选用于毛状根侵染的外植体提高毛状根诱导率,通过筛选毛状根的愈伤组织诱导培养基和不定芽诱导培养基优化毛状根的植株再生效率,最终建立了菠菜毛状根遗传转化体系和菠菜毛状根再生体系,为菠菜基因功能研究奠定基础。主要研究结果如下:1.首先,为了提高菠菜毛状根诱导效率,对用于侵染的外植体进行筛选。选择了菠菜Sp75、Sp73的叶片和叶柄作为外植体,对毛状根诱导效率进行统计。发现Sp75叶柄的诱导效率最高,可达到22.3%;Sp75叶片仅为15.7%;Sp73叶柄和叶片的毛状根诱导率分别为10.3%和14%。2.通过在愈伤诱导培养基添加NAA、GA₃激素,使得以毛状根为外植体诱导得到的愈伤组织具有胚性。通过在不定芽诱导培养基中添加6-BA、NAA、KT等激素,使以菠菜毛状根作为外植体的不定芽诱导率提高到46.7%。3.利用CRISPR/Cas9技术构建了So MBF1s敲除载体,通过菠菜毛状根遗传转化体系,成功获得So MBF1s纯合敲除突变体毛状根。mbf1c纯合突变类型包括单个A碱基插入和4个碱基缺失两种类型,mbf1b纯合突变类型包括单个C碱基缺失和4个碱基缺失两种类型。同时,利用已构建的p Cambia2300-So MBF1s-GFP载体,通过菠菜毛状根遗传转化体系,成功获得So MBF1s过表达毛状根。q RT-PCR检测So MBF1s过表达毛状根的表达量,获得5个So MBF1c过表达毛状根和5个So MBF1b过表达毛状根,其中So MBF1b表达量最高的株系OE-7约为对照毛状根的44倍,So MBF1c表达量最高的株系OE-16约为对照毛状根的45倍。4.为了更好探究So MBF1c在耐热方面的功能,对液体培养基培养10天的So MBF1c过表达毛状根、So MBF1c敲除纯合毛状根和对照毛状根,进行39℃高温处理24h。发现So MBF1c过表达毛状根的鲜重比对照毛状根的更高,而So MBF1c敲除纯合毛状根的鲜重却较对照毛状根更低。So MBF1c过表达毛状根热处理后H₂O₂含量较对照毛状根高,而敲除纯合毛状根的H₂O₂含量较对照毛状根低,表明So MBF1c能提高菠菜毛状根的耐热性。5.通过对17个物种中191种TPS蛋白进行系统发育树分析,发现TPS蛋白分为两类。菠菜有11个成员,将其主要分为两类,So TPS1-3、So TPS9-11属于Ⅰ类,So TPS4-8属于Ⅱ类。6.克隆了菠菜So TPSs基因家族中6个成员。基因结构分析发现,所有Ⅰ类So TPSs基因的内含子数量比Ⅱ类So TPSs基因内含子的数量多,Ⅱ类So TPSs基因内含子数量不超过3个,外显子数量都是3个;保守基序分析发现,II类成员拥有所有10个保守基序,而I类成员则有所缺失;染色体定位分析发现,So TPS2和So TPS3都位于4号染色体上,So TPS6和So TPS10位于3号染色体,而其他染色体上仅存在单个基因;顺式作用元件分析发现,So TPSs基因可能会在应激、激素、光反应和生长发育等方面中发挥了重要作用。上述研究结果为后续进行菠菜基因功能研究提供了基础。更多还原

【Abstract】 Spinach（Spinacia oleracea L.）is a cold-resistant but heat-resistant leafy vegetable.The growth,yield and quality of spinach are seriously affected by high temperature stress.Identifying key heat-tolerant genes in spinach will help in developing heat-tolerant spinach varieties using molecular breeding strategies.However,the lack of an efficient genetic transformation system in spinach has limited the functional identification and in-depth study of heat-tolerant genes.We screened explants used for hairy roots induction to improve hairy root in duction efficiency.Callus induction medium and adventitious bud induction medium of hairy roots were also optimized.The genetic transformation system of spinach hairy roots and the regeneration system of spinach hairy roots were optimized,which laid a foundation for the study of spinach gene function.The main findings are as follows:1.Firstly,the explants were screened to improve the induction efficiency of spinach hairy roots.The leaves and petioles of Sp75 and Sp73 were used as the infected explants,and the hairy root induction efficiency was counted.The highest induction efficiency of Sp75 petiole was 22.3%,whereas Sp75 leaves were only 15.7%.The hairy root induction efficiency of Sp73 petiole and leaf were 10.3%and 14%.2.NAA and GA3 were added to the medium,the callus tissue induced from hairy roots acquired embryogenic characteristics.With the addition of hormones such as 6-BA,NAA,and KT to the shoot induction medium,the induction rate of adventitious shoots using spinach hairy roots as explants was increased to 46.7%.3.The So MBF1s knockout vector was constructed by CRISPR/Cas9 technology.Through the genetic transformation system of spinach hairy roots,So MBF1s homozygous knockout mutant hairy roots were successfully obtained.The homozygous mutation types of mbf1c included insertion of one A base and deletion of four bases,and the homozygous mutation types of mbf1b were deletion of one C base and deletion of four bases.At the same time,So MBF1s overexpressed hairy roots were successfully obtained using the constructed p Cambia2300-So MBF1s-GFP vector through the genetic transformation system of spinach hairy roots.The expression level of So MBF1s overexpressing hairy roots was detected by q RT-PCR.Five So MBF1c overexpressing hairy roots lines and five So MBF1b overexpressing hairy roots lines were found.Among them,OE-7 was the highest expression level of So MBF1b and was about 44times of the control hairy roots,OE-16 was the highest expression level of So MBF1c and was about 45 times of the control hairy roots.4.In order to better explore the function of So MBF1c in heat resistance,So MBF1c overexpression hairy roots,mbf1c homozygous hairy roots and control hairy roots were cultured in liquid medium for 10 days and treated at 39°C for 24 hours.It was found that overexpression of So MBF1c resulted in a higher fresh weight of hairy roots compared to the control,while the fresh weight of homozygous hairy roots with So MBF1c knocked out was lower than that of the control.After heat treatment of the hairy roots,the H2O2 content in the hairy roots with So MBF1c overexpression was higher than that in the control,while the H₂O₂ content in the homozygous hairy roots with So MBF1c knocked out was lower than that in the control,indicating that So MBF1c can improve the heat tolerance of spinach hairy roots.5.Phylogenetic tree analysis was used to analyze 191 TPS proteins in 17 species.It was found that TPS proteins were divided into two categories.Spinach contained 11members,which were divided into two categories.Class I included So TPS1-3,So TPS9-11,and Class II included So TPS4-8.6.Six members of spinach So TPSs gene family were cloned.Through the gene structure analysis,the introns number of all class I was more than class II,the introns number of class II was no more than three,while the exons number was three.Conservative motif analysis found that class II members all have 10 conserved motifs,while class I members are deletion.Chromosome localization analysis,found that So TPS2 and So TPS3 were found on chromosome 4,So TPS6 and So TPS10 were found on chromosome 3,while only a single gene existed on other chromosomes.Cis-acting element analysis found that So TPSs genes may play an important role in stress,hormones,light response and growth and development.These results provided the basis for further studies on gene function of spinach.更多还原