CN117384950A - MsSPL17基因的应用 - Google Patents
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Abstract
本发明公开了一种MsSPL17基因的应用，涉及生物技术技术领域，包括MsSPL17基因在S1）增加紫花苜蓿的分枝数中的应用；S2）在缩短紫花苜蓿节间长度的应用；S3）在增加紫花苜蓿叶片数目的应用；S4）在增加紫花苜蓿的产量中的应用；S5）在制备多分枝数的紫花苜蓿产品中的应用；S6）在培育多分枝数的转基因紫花苜蓿中的应用。通过改变MsSPL17基因的表达可以调控紫花苜蓿分枝数的多少，进而调控紫花苜蓿的节间长度、叶片数、产量等。
Description
技术领域
本发明属于生物技术领域，涉及一种MsSPL17基因的应用。
背景技术
对于紫花苜蓿而言，产量决定了紫花苜蓿作为优质牧草的应用程度，而叶量是产量的基础，分枝数是叶量的基础，分枝数与产量的相关系数在0.6以上，则在既定范围阈值内紫花苜蓿的分枝数就越多，产量也越高。因此分枝数的选择在紫花苜蓿育种中具有举足轻重的地位。
把分枝数作为育种目标进行作物改良已在多个物种中应用，作为紫花苜蓿重要的产量因子，利用重要的分枝数基因选择或调整紫花苜蓿产量尤为重要。
发明内容
为实现紫花苜蓿的产量的选择和调控，本发明提供了一种通过应用MsSPL17基因或MsSPL17蛋白调控紫花苜蓿分枝数、节间长度以及叶片数的方法，从而提高紫花苜蓿的产量和品质。
为实现本发明的目的，本发明第一方面提供一种MsSPL17基因的应用，通过沉默紫花苜蓿中的MsSPL17基因实现，为S1）或S2）或S3）或S4）或S5）或S6）；
S1）在调控紫花苜蓿的分枝数中的应用；
S2）在调控紫花苜蓿节间长度的应用；
S3）在调控紫花苜蓿叶片数目的应用；
S4）在调控紫花苜蓿的产量中的应用；
S5）在制备多分枝数的紫花苜蓿产品中的应用；
S6）在培育多分枝数的转基因紫花苜蓿中的应用。
具体的，所述MsSPL17基因的gDNA序列如SEQ ID NO.1 所示；
具体的，所述MsSPL17基因的CDS序列如SEQ ID NO.2 所示；
具体的，所述MsSPL17蛋白的氨基酸序列如SEQ ID NO.3 所示。
具体的，所述MsSPL17基因的CDS编码序列通过如SEQ ID NO.4-5所示的引物对扩增获得：
SEQ ID NO.4 CDS-F: ATGGAGTGGAGTAATTTG
SEQ ID NO.5 CDS-R： CTAATCCCATTGAAAAGGA。
尤其是，检测所述MsSPL17基因在植物中表达量的引物如SEQ ID NO.6-7所示：
SEQ ID NO.6 SPL17-RT-F: GAACCAAGTCCTGAATTGAATAAC
SEQ ID NO.7 SPL17-RT-R: CATCCATATAATGCCTCCTTCC。
尤其是，所述具有过表达载体pbi121-△GUS的载体臂的MsSPL17基因的编码序列通过如SEQ ID NO.8-9所示的引物对扩增获得：
SEQ ID NO.8 p121-F: acacgggggactctagaATGGAGTGGAGTAATTTG
SEQ ID NO.9 p121-R: gggactgaccacccgggCTAATCCCATTGAAAAGGA。
尤其是，所述具有RNAi沉默载体的载体臂的MsSPL17基因片段通过如SEQ IDNO.10-11所示的引物对扩增得到：
SEQ ID NO.10：attB1SPL17:GGGGACAAGTTTGTACAAAAAAGCAGGCTTGAAGGATGTAAACTAG
SEQ ID NO.11：attB2SPL17:GGGGACCACTTTGTACAAGAAAGCTGGGTCAAAACGTGAGGTCAAG。
尤其是，鉴定是否为所述MsSPL17基因的过表达转基因阳性植株的引物对如SEQID NO.12-13所示：
SEQ ID NO.12 p121seq-F: CGTAAGGGATGACGCACA
SEQ ID NO.13 p121seq-R: GGGTTTTCCCAGTCACGA。
尤其是，鉴定是否为所述MsSPL17基因的基因沉默转基因阳性植株的引物对如SEQID NO.14-15所示：
SEQ ID NO.14 attBseq-F: TGAAGGATGTAAACTAG
SEQ ID NO.15 attBseq-R: ACTTCCTACATTTGATC。
为实现本发明技术目的，本发明第二方面提供MsSPL17蛋白在增加紫花苜蓿分枝数的应用，或在增加紫花苜蓿产量的应用。
为实现本发明技术目的，本发明第二方面提供一种调节剂，其具有MsSPL17基因或MsSPL17蛋白，使紫花苜蓿的分枝数、叶片数、产量增加或缩短紫花苜蓿的节间长度。
附图说明
图1 是MsSPL17过表达植株DNA水平鉴定结果，其中a,b,c代表DNA Marker; CK1及CK2代表对照植株；其余数字代表获得的再生植株；
图2 是MsSPL17的RNAi沉默植株DNA水平鉴定，其中，a代表DNA Marker; 17代表构建的重组载体; CK1代表对照植株;其余数字代表获得的再生植株；
图3 是MsSPL17过表达及RNAi沉默转基因紫花苜蓿表型。
具体实施方式
下面参考具体实施例，对本发明进行说明，需要说明的是，这些实施例仅仅是说明性的，而不能理解为对本发明的限制。若未特别指明，实施例中所采用的技术手段为本领域技术人员所熟知的常规手段，所采用的试剂和产品也均为可商业获得的。未详细描述的各种过程和方法是本领域中公知的常规方法，所用试剂的来源、商品名以及有必要列出其组成成分者，均在首次出现时标明，其后所用相同试剂如无特殊说明，均以首次标明的内容相同。
实施例1 紫花苜蓿MsSPL17基因的克隆
1、紫花苜蓿MsSPL17基因序列信息的获得
以拟南芥SPL基因保守序列为query序列，Blast检索 Alfalfa CADL GenomeAssembly V1和Alfalfa CADL Annotation V1 CDS，获得紫花苜蓿MsSPL17基因的序列信息。
2、MsSPL17基因的克隆
根据获得的紫花苜蓿MsSPL17基因的序列信息设计扩增引物CDS-F:ATGGAGTGGAGTAATTTG以及CDS-R：CTAATCCCATTGAAAAGGA，分别提取紫花苜蓿品种“中苜1号”的RNA以及DNA，以反转录的cDNA和gDNA为模板分别进行PCR扩增，扩增后进行琼脂糖凝胶电泳，回收扩增产物并测序，利用spidey软件比对获得的紫花苜蓿MsSPL17基因的基因组序列以及cDNA序列，同时获得该基因的基因结构，即由三个外显子及两个内含子组成，MsSPL17基因的核苷酸序列如SEQ ID NO.1所示，由该基因编码的MsSPL17蛋白的序列如SED IDNO.2所示，该基因的CDS序列如SED ID NO.3所示。
实施例2、紫花苜蓿MsSPL17基因时空表达模式分析
1、紫花苜蓿不同组织样品cDNA获得
于穴盆中播种“中苜一号”种子，待苜蓿苗地上部分长至15cm高时收集茎、叶片、根、颈部组织至于液氮中速冻，保存于-80℃待用，剩余植株继续生长于穴盆中；第二年春苜蓿返青后，收集返青期、分枝期、现蕾期、开花期以及结荚期的根、茎、茎节、叶、花、蕾及荚果组织液氮中速冻，保存于-80℃待用。使用PROMEGA Eastep Super RNA提取试剂盒提取上述组织的RNA，经琼脂糖凝胶电泳检测RNA质量，经Nanodrop检测RNA数量后，利用TransScriptAll-in-One First-Strand cDNA Synthesis SuperMix for qPCR反转录试剂盒合成cDNA，将此cDNA稀释4倍，以作模板，合成反应及反应条件如下：
2、紫花苜蓿MsSPL17基因组织表达模式分析
根据已获得的紫花苜蓿MsSPL17基因序列信息，利用AlleleID 6.0设计荧光定量引物SPL17-RT-F: GAACCAAGTCCTGAATTGAATAAC以及SPL17-RT-R:CATCCATATAATGCCTCCTTCC,以紫花苜蓿不同组织cDNA为模板，使用TB Green™ Premix Ex Taq ™II (Tli RNaseH Plus)试剂，ABI Q7型荧光定量PCR仪进行qRT-PCR扩增，扩增反应体系如下：
利用2-ΔΔCt法计算MsSPL17基因在紫花苜蓿不同组织中的相对表达量，如表1所示：
表1 MsSPL17基因在紫花苜蓿不同组织中的相对表达量
根据表1的分析结果可知，MsSPL17基因在分枝期表达量较高，尤其在植物顶端组织、茎节组织，可见MsSPL17基因参与了紫花苜蓿的分枝的发育，从而影响紫花苜蓿的分枝数。
实施例3、MsSPL17基因过表达及RNAi沉默转基因紫花苜蓿获得
1、过表达MsSPL17转基因载体构建
使用限制性内切酶XbaI及XmaI双酶切植物过表达载体pbi121-△GUS，同时利用引物p121-F: acacgggggactctagaATGGAGTGGAGTAATTTG以及p121-R: gggactgaccacccgggCTAATCCCATTGAAAAGGA扩增MsSPL17的CDS区，以获得包含载体臂的MsSPL17基因的CDS区，利用LanGene™Seamless Cloning&Assembly Kit（无缝克隆试剂盒）将载体和片段重组，重组产物转化DH5a大肠杆菌感受态，经测序验证获得过表达MsSPL17的转基因载体pbi121-△GUS::MsSPL17。
2、RNA沉默的MsSPL17转基因载体构建
利用引物对attB1SPL17:GGGGACAAGTTTGTACAAAAAAGCAGGCTTGAAGGATGTAAACTAG/attB2SPL17:GGGGACCACTTTGTACAAGAAAGCTGGGTCAAAACGTGAGGTCAAG扩增MsSPL17的CDS区，以获得包含attB的244bp的MsSPL17片段，利用BP反应将attBSPL17连接至pDONR221载体，转化至TOP10大肠杆菌感受态细胞，经转化及质粒测序后获得中间载体pENTER-MsSPL17，再次利用LR反应将该片段转移至RNAi载体pK7GWIWG2D(II)上，经测序验证获得MsSPL17的RNAi沉默载体pK7GWIWG2D(II)::MsSPL17。
3、MsSPL17过表达及RNAi沉默的转基因紫花苜蓿植株获得
将构建的过表达载体pbi121-△GUS::MsSPL17及RNAi沉默载体pK7GWIWG2D(II)::MsSPL17转化农杆菌GV3101感受态，挑取阳性克隆培养至50ml，利用农杆菌介导的叶圆盘法转化紫花苜蓿品种“中苜1号”，经愈伤组织诱导及再生获得再生植株。提取再生植株的基因组DNA,分别使用引物对p121seq-F: CGTAAGGGATGACGCACA/p121seq-R:GGGTTTTCCCAGTCACGA，以及引物对attBseq-F:TGAAGGATGTAAACTAG/attBseq-R:ACTTCCTACATTTGATC，对再生植株进行DNA水平的鉴定，阳性植株即为获得的MsSPL17过表达（图1）及基因沉默（图2）的转基因紫花苜蓿。
4、转基因紫花苜蓿MsSPL17表达量分析
使用PROMEGA Eastep Super RNA提取试剂盒提取MsSPL17过表达及RNAi沉默的紫花苜蓿RNA，经琼脂糖凝胶电泳检测RNA质量，经Nanodrop检测RNA数量后，利用HiScript®III All-in-one RT SuperMix Perfect for qPCR反转录试剂盒合成cDNA，将此cDNA稀释4倍，作为模板，使用引物SPL17-RT-F: GAACCAAGTCCTGAATTGAATAAC/SPL17-RT-R:CATCCATATAATGCCTCCTTCC，定量试剂Taq Pro Universal SYBR qPCR Master MixqRT-PCR，ABI Q7型荧光定量PCR仪进行qRT-PCR扩增检测阳性植株中MsSPL17的表达量，利用2-ΔΔCt法计算目的基因相对表达水平。结果如表2所示，编号为OE25的过表达植株中的MsSPL17表达量最高，是对照植株的180倍；编号为RNA11的RNAi沉默植株中的MsSPL17表达量最低，是对照植株的0.024倍。
表2 部分转基因紫花苜蓿中MsSPL17的表达量
实施例4、MsSPL17基因过表达及基因沉默转基因紫花苜蓿表型分析
选择CK1以及表达量较高的转基因紫花苜蓿扦插后，选择长势一致的植株进行统一管理后的表型观察发现，MsSPL17过表达转基因植株在分枝数、节间长度、株高及生物量性状间不存在显著性差异（如图3所示的部分植株表型照片）；而MsSPL17基因沉默的转基因植株分枝数显著高于对照植株，此外，RNA沉默的植株还表现出节间长度缩短、生物量增加等表型，而在株高性状上则不存在显著性差异（图3）。以上表型性状的显著差异表明在紫花苜蓿中沉默MsSPL17可增加紫花苜蓿的分枝数，缩短节间长度，增加叶片数目，进而增加紫花苜蓿的产量。
本发明的内容不限于具体实施例所举例，本领域技术人员通过阅读本说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。
Claims (10)
1.MsSPL17基因的应用，其特征在于，通过沉默紫花苜蓿中的MsSPL17基因实现，为S1）或S2）或S3）或S4）或S5）或S6）：
S1）增加紫花苜蓿的分枝数中的应用；
S2）在缩短紫花苜蓿节间长度的应用；
S3）在增加紫花苜蓿叶片数目的应用；
S4）在增加紫花苜蓿的产量中的应用；
S5）在制备多分枝数的紫花苜蓿产品中的应用；
S6）在培育多分枝数的转基因紫花苜蓿中的应用。
2.如权利要求1所述的MsSPL17基因的应用，所述MsSPL17基因的gDNA序列如SEQ IDNO.1 所示；
所述MsSPL17基因的CDS序列如SEQ ID NO.2 所示；
所述MsSPL17蛋白的氨基酸序列如SEQ ID NO.3 所示。
3.如权利要求1所述的MsSPL17基因的应用，所述MsSPL17基因的CDS编码序列通过如SEQ ID NO.4-5所示的引物对扩增获得。
4.如权利要求1所述的MsSPL17基因的应用，检测所述MsSPL17基因在植物中表达量的引物如SEQ ID NO.6-7所示。
5.如权利要求1所述的MsSPL17基因的应用，具有所述MsSPL17基因的过表达载体pbi121-△GUS的载体臂的编码序列通过如SEQ ID NO.8-9所示的引物对扩增获得。
6.如权利要求1所述的MsSPL17基因的应用，具有所述MsSPL17基因片段的RNAi沉默载体的载体臂的编码序列通过如SEQ ID NO.10-11所示的引物对扩增得到。
7.如权利要求1所述的MsSPL17基因的应用，鉴定是否为所述MsSPL17基因的过表达转基因阳性植株的引物对如SEQ ID NO.12-13所示。
8.如权利要求1所述的MsSPL17基因的应用，鉴定是否为所述MsSPL17基因的基因沉默转基因阳性植株的引物对如SEQ ID NO.14-15所示。
9.MsSPL17蛋白在增加紫花苜蓿分枝数、叶片数、产量或缩短紫花苜蓿节间长度的应用。
10.一种调节剂，其特征在于，其具有MsSPL17基因或MsSPL17蛋白，使紫花苜蓿的分枝数、叶片数、产量增加或缩短紫花苜蓿的节间长度。
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