CN114561421B - 石龙芮体细胞胚介导的遗传转化方法 - Google Patents
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Abstract
本发明属于生物技术领域，具体涉及石龙芮体细胞胚介导遗传转化的方法。本发明可快速、高效诱导出石龙芮体细胞胚结构，可以通过“外植体侵染‑体细胞胚诱导”方法成功转化外源基因，所建立的石龙芮体细胞胚介导的遗传转化体系具有快速、高效、嵌合率低、可稳定遗传等特点。
Description
技术领域
本发明属于生物技术领域，具体涉及石龙芮体细胞胚介导的遗传转化方法。
背景技术
石龙芮是一种毛茛科中草药，富含药用价值较高的次生代谢物，包括生物碱、单宁、黄酮类化合物、异黄酮和原儿茶醛。长期以来，石龙芮一直被中国传统医学用来防止乙型肝炎病毒HBV和单纯疱疹病毒1型HSV-1的复制，其还可用于治疗黄疸、风湿性疼痛、哮喘和小便失禁。同时，石龙芮也是控制黑腹果蝇和赤拟谷盗等害虫非常有效的杀虫剂。此外，石龙芮具有较强的污水处理能力，能吸收大量氮、磷，积累和监测土壤中重金属的含量变化，如铜、铅、铁、锌等。
现有技术对毛茛科植物的研究多集中于该科植物的次生代谢物质的活性检测、毒性与活性成分组分分析，土壤重金属与有机污染物的清除与净化，以及二倍体黑种草、耧斗菜等观赏性毛茛科植物花器官的发育机制等方面。多种毛茛科植物的常规无菌苗培养过程尚且存在一些技术难度，如种子萌发率、发芽势的同步率、无菌苗的继代培养等。关于毛茛科植物体细胞胚发生及其介导的高效再生、常规再生体系以及遗传转化体系均无可参考的现行体系。石龙芮体细胞胚介导的高效再生与遗传转化等体系建立存在诸多技术瓶颈：如培养基筛选、种子催芽、植物生长调节剂种类与浓度筛选、最佳外植体的筛选以及农杆菌侵染时段的选择等。因此，石龙芮体细胞胚介导的高效再生与遗传转化体系方面未有研究报道。
发明内容
本发明的目的在于提供石龙芮体细胞胚介导遗传转化的方法。
根据本发明具体实施方式的石龙芮体细胞胚介导遗传转化的方法，所述方法包括以下步骤：
(1)将石龙芮种子消毒，播于MS培养基中，进行无菌苗培养，其中MS培养基包括以下组分：MS盐、B5有机、30g/L蔗糖、7.8g/L琼脂粉，MS培养基的pH为5.8；
(2)选择无菌苗的营养器官为外植体，在转化侵染介质中进行农杆菌转化介质侵染；
(3)将侵染后的外植体平铺于共培养培养基中进行共培养；
(4)将共培养后的外植体转入含有抗生素的体细胞胚诱导培养基中进行体细胞胚的诱导发生以及抗性体细胞胚的筛选，其中，体细胞胚诱导培养基包括以下组分：MS盐、B5有机、10.0mg/LNAA、30g/L蔗糖、3.6g/L结冷胶，体细胞胚诱导培养基的pH为5.8；
(5)将抗性体细胞胚置于生根培养基中，于光照条件下诱导胚体生根，其中，生根培养基包括以下组分：1/2MS盐、1/2B5有机、0.1mg/LNAA、15g/L蔗糖、7.8g/L琼脂粉，生根培养基的pH为5.8；
(6)炼苗、移栽。
根据本发明具体实施方式的石龙芮体细胞胚介导遗传转化的方法，步骤(2)中，转化侵染介质包括以下组分：MS盐、B5有机、15g/L葡萄糖，转化侵染介质的pH为5.8。
根据本发明具体实施方式的石龙芮体细胞胚介导遗传转化的方法，步骤(3)中，共培养培养基包括以下组分：MS盐、B5有机、40mg/L乙酰丁香酮、30g/L蔗糖、7.8g/L琼脂粉，共培养培养基的pH为5.8。
根据本发明具体实施方式的石龙芮体细胞胚介导遗传转化的方法，步骤(4)中，体细胞胚诱导培养基含有100mg/L硫酸卡那霉素和500mg/L羧苄青霉素钠。
根据本发明具体实施方式的石龙芮体细胞胚介导遗传转化的方法，步骤(5)中，将抗性体细胞胚置于25℃，16h光照/8h黑暗，光照度为120μmol.m-2s-1条件下诱导生根。
本发明的有益效果：
本发明可快速、高效诱导出石龙芮体细胞胚结构，可以通过“外植体侵染-体细胞胚诱导”方法成功转化外源基因，所建立的石龙芮体细胞胚介导的遗传转化体系具有快速、高效、嵌合率低、可稳定遗传等特点。
附图说明
图1显示石龙芮体细胞胚不同的发育阶段；其中，A为石龙芮叶片外植体诱导出的多个体细胞胚结构；B为球形胚；C为球形-心形过渡胚；D、E、F和G为心形胚；H为鱼雷胚；I、J、K和L为子叶胚；
图2显示体细胞胚介导的石龙芮再生情况；其中，A为由石龙芮叶片外植体诱导出的不同发育阶段的体细胞胚结构；B为体细胞胚发育流程，显示具有不同子叶数的子叶胚；C为成熟胚；D为体细胞胚生根诱导；E为体细胞胚再生苗；F为石龙芮再生植株；F1和F2为石龙芮再生植株花器官；
图3显示过表达RcLEC1-B导致石龙芮的矮化与花器官畸形；其中，A、A1、A2和B为石龙芮遗传转化过程，由根、茎、叶诱导出处于不同阶段的硫酸卡那霉素(Kan+)阳性胚体；A为石龙芮根外植体；A1为石龙芮茎外植体；A2和B为石龙芮叶外植体；C1为石龙芮阳性胚体的GUS染色；C2为石龙芮阳性花器官的GUS染色；D和D1为石龙芮野生植株(15d)；E和E1为石龙芮空载植株(pCAMBIA2300，15d)；D2为石龙芮野生植株(30d)；E2为石龙芮空载植株(pCAMBIA2300，30d)；F和F1为具有矮化表型的RcLEC1-B-OE石龙芮植株(15d)；F2为RcLEC1-B-OE石龙芮植株(30d)；G为具有角质层缺陷表型的RcLEC1-B-OE石龙芮植株(15d)；
图4显示RcLEC1-B-OE石龙芮与野生型植株的花器官形态与数量，其中，A具有5个萼片、5个花瓣和16个雄蕊和1个总雌蕊；B-B16为RcLEC1-B-OE石龙芮花器官；B、B2、B3、B4、B5、B6、B7和B10为RcLEC1-B-OE石龙芮花器官中具有畸形的花瓣状结构；B4为RcLEC1-B-OE石龙芮花器官中具有介于花瓣与萼片的过渡态结构；B-B16为不具有花瓣与雄蕊结构的RcLEC1-B-OE石龙芮花器官；
图5显示石龙芮根、茎和叶的体细胞胚诱导发生情况，其中，A-C为发育早期的石龙芮体细胞胚；A1-C1为发育晚期的石龙芮体细胞胚；A和A1为根外植体；B和B1为茎外植体；C和C1为叶外植体。
具体实施方式
本发明所用培养基见表1：
表1本发明所用培养基名称缩写、名称与组分
实施例1
(1)无菌苗培养：选择健康饱满的石龙芮种子，先用75％(v/v)乙醇进行表面消毒30s-1min，无菌水冲洗3-5遍；再用2.5％次氯酸铵消毒处理8-10min，无菌水冲洗3-5遍；将消毒后的种子播于含有改良MS培养基的组培瓶中，在低温(4℃)条件下催芽2-3d，然后于黑暗条件下，孵育种子(25±1℃)直至种壳露白。
将培养皿于25℃，16h光照/8h黑暗，光照度为120μmol.m-2s-1的组织培养条件下进行培养。
(2)待石龙芮组培苗生长至4-5片真叶，选取石龙芮的根、茎(无腋芽)和叶三种外植体，置于体细胞胚诱导培养基SE-M(Somatic embryogenesis induction medium)上，进行体细胞胚结构的诱导。
如图1所示，石龙芮体细胞胚的发育属于经典SE路径，历经原胚-球形胚-心形胚-鱼雷胚-子叶胚。
(3)将诱导出的石龙芮体细胞胚转入生根培养基RO-M(Rooting medium)，进行垂直板培养，诱导胚体生根、成苗。
如图2所示，单个胚体可诱导出正常根系，胚体根系诱导率为100％，并可形成单株实生小苗；经过培养可正常发育、开花。
实施例2
(1)无菌苗培养：选择健康饱满的石龙芮种子，先用75％(v/v)乙醇进行表面消毒30s-1min，无菌水冲洗3-5遍；再用2.5％次氯酸铵消毒处理8-10min，无菌水冲洗3-5遍；将消毒后的种子播于含有改良MS培养基的组培瓶中，在低温(4℃)条件下催芽2-3d，然后于黑暗条件下，孵育种子(25±1℃)直至种壳露白；
将培养皿于25℃，16h光照/8h黑暗，光照度为120μmol.m-2s-1的组织培养条件下进行培养；
(2)将石龙芮的根、茎(无腋芽)和叶三种外植体先进行农杆菌转化介质SU-M(Suspensions medium)侵染，期间不断摇动8-10min，使外植体与转化介质充分接触；
将侵染后的外植体从转化介质中取出，先用无菌滤纸吸去多余的转化介质，平铺于培养基CC-M(Co-culture medium)中进行共培养，然后将之置于25℃的恒温培养箱，进行共培养处理72h；
(3)将共培养后的石龙芮外植体转入体细胞胚发生-筛选培养基SEIS-M中进行体细胞胚的诱导发生以及抗性体细胞胚的筛选。
如图3所示，根、茎、叶三种外植体均可诱导出抗性体细胞胚结构，以叶片外植体为最佳受体外植体。GUS染色结果显示，胚体可X-gluc底物存在的情况下，被染成了蓝色，如图3中C1，阳性苗的单花亦可被染色成蓝色，如图3中C2。
(4)将抗性体细胞胚置于生根培养基RO-M，于光照条件下(25℃，16h光照/8h黑暗，光照度为120μmol.m-2s-1)诱导胚体生根、成苗；
(5)将由体细胞胚诱导出来的抗性芽开瓶炼苗7d，用清水小股水流清洗掉根系所残留的琼脂块，将石龙芮小苗移栽至培养介质(大蛭石:花卉土＝1:1)中，于弱光下进行保鲜膜保湿培养。15d后除去保鲜膜，在较为湿润的环境中进行养护。
实施例3
(1)将石龙芮叶片外植体进行农杆菌转化介质SU-M(RcLEC1-B-OE)侵染，摇动8-10min；将侵染后的外植体平铺于共培养培养基CC-M，25℃黑暗共培养处理72h；
(2)共培养后的石龙芮叶片外植体转入体细胞胚发生-筛选培养基SEIS-M进行抗性体细胞胚的筛选；
(3)将石龙芮抗性体细胞胚置于生根培养基RO-M诱导胚体生根、成苗；
(4)通过上述转基因方法，过表达RcLEC1-B同样导致了石龙芮植株的矮化(图3中F、F2)、叶片畸形(图3中F1和G)、花器官的畸化；其中，如图4所示，花器官的畸化主要表现为花瓣、萼片、雄蕊的个数变化与形态变化。该结果RcLEC1-B可导致转基因拟南芥矮化与花器官畸形等研究结果相一致。
将外植体置于体细胞胚诱导培养基SE-M上进行体细胞胚结构诱导的过程中，考察不同浓度NAA对石龙芮根、茎、叶外植体诱导体细胞胚的影响，结果如表2、图5所示。
表2不同浓度NAA对石龙芮根、茎、叶外植体诱导体细胞胚的影响
注：采用SPSS16.0进行Duncan检验，对30个培养皿300个外植体计算产生体细胞胚个数的平均值和标准误差。大写字母和小写字母分别表示1％和5％概率水平的显著差异。
如图5所示，纵向分析不同浓度NAA对石龙芮根、茎、叶外植体诱导体细胞胚的影响，10mg/LNAA的诱导产胚率最高，明显高于0mg/L、1.0mg/L、2.5mg/L、5.0mg/L、20.0mg/L等浓度。根据表2数据，横向分析三种不同外植体的诱导效果，叶片为最佳胚体诱导外植体，单个外植体可产生213.63个胚体，远大于根外植体产生的66.03个以及茎外植体产生的126.47个。
Claims (1)
1.石龙芮体细胞胚介导的遗传转化方法，其特征在于，所述方法包括以下步骤：
(1)将石龙芮种子消毒，播于无菌苗培养基中，进行无菌苗培养，其中，所述菌苗培养基的配方为：MS盐、B5有机、30g/L蔗糖、7.8g/L琼脂粉，pH5.8，在4℃条件下催芽2-3d，然后于黑暗条件下，25±1℃孵育种子直至种壳露白，然后于25℃，16h光照/8h黑暗，光照度为120μmol.m-2s-1的组织培养条件下进行培养；
(2)选择无菌苗的根、无腋芽的茎或叶作为外植体，在转化侵染介质中进行农杆菌转化介质侵染，其中，所述转化侵染介质为MS盐、B5有机、15g/L葡萄糖，pH为5.8；共培养培养基为MS盐、B5有机、40mg/L乙酰丁香酮、30g/L蔗糖、7.8g/L琼脂粉，pH为5.8；
(3)将共培养后的石龙芮外植体转入体细胞胚发生-筛选培养基SEIS-M中进行体细胞胚的诱导发生以及抗性体细胞胚的筛选，所述体细胞胚发生-筛选培养基SEIS-M为MS盐、B5有机，40mg/L乙酰丁香酮、10mg/LNAA、100mg/L硫酸卡那霉素、500mg/L羧苄青霉素钠、30g/L蔗糖、7.8g/L琼脂粉，pH为5.8；
(4)将共培养后的外植体转入含有抗生素的体细胞胚诱导培养基中进行体细胞胚的诱导发生以及抗性体细胞胚的筛选，其中，体细胞胚诱导培养的配方为：MS盐、B5有机、10.0mg/LNAA、30g/L蔗糖、7.8g/L琼脂粉，100mg/L硫酸卡那霉素和500mg/L羧苄青霉素钠，体细胞胚诱导培养基的pH为5.8；
(5)将抗性体细胞胚置于生根培养基中，于25℃，16h光照/8h黑暗，光照度为120μmol.m-2s-1条件下诱导生根，其中，生根培养基的配方为：1/2MS盐、1/2B5有机、0.1mg/LNAA、15g/L蔗糖、7.8g/L琼脂粉，生根培养基的pH为5.8；
(6)炼苗、移栽。
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