摘要
以枸杞品种‘宁杞1号’花药为材料,采用RT-PCR技术,分离了R2R3类MYB基因LbMYB103包含完整开放阅读框(ORF)的cDNA片段,碱基序列与已知基因HQ415755完全一致。运用Gateway技术构建LbMYB103基因植物过表达载体pMDC83-LbMYB103,利用基因枪法将融合有绿色荧光蛋白(GFP)的过表达载体转入洋葱表皮细胞,将LbMYB103基因定位在细胞核。实时荧光定量PCR分析发现,LbMYB103基因在花药中优势表达,果实中表达量较低,在根、茎和叶中均未检测到其转录本,推测LbMYB103基因可能在花药发育过程中起重要作用。通过根癌农杆菌介导法将pMDC83-LbMYB103转入拟南芥(Col-0),经筛选获得T1代抗性再生植株52棵,PCR鉴定有41棵阳性植株,收获T1代种子,经抗性筛选获得T2代抗性植株29棵,PCR鉴定有23棵阳性植株。实时荧光定量PCR分析表明,LbMYB103在拟南芥植株的基因组中正常表达。表型观察发现T1和T2代拟南芥花药发育异常,花发育迟缓,果荚短小无种子,进一步表明LbMYB103可能与植物的育性有关。该结果为进一步开展枸杞遗传转化,深入研究LbMYB103基因在枸杞花药发育过程中可能发挥的调控功能奠定了基础。
Using RT-PCR technology,we cloned the R2R3 MYB gene LbMYB103 cDNA fragment,which contains a complete open reading frame（ORF）,from wolfberry variety ‘Ningqi No.1＇.The base sequence was entirely consistent with that of the known gene HQ415755.The plant over expression vector pMDC83-LbMYB103-GFP was constructed with Gateway Technology and confirmed by enzyme digestion and sequencing.We transformed pMDC83-LbMYB103 into onion epidermal cells using particle bombardment and found that LbMYB103 was located in the nucleus.We used the real-time quantitative PCR approach to analyze the expression pattern of LbMYB103 in various organs,and the result showed that expression of LbMYB103 was the highest in flower and anther,relatively lower in fruits,but undetectable in leaf,stem and root,indicating that LbMYB103 may play some roles in wolfberry reproductive organ process,such as anther development.We transformed pMDC83-LbMYB103 into Arabidopsis thaliana（Col-0）using Agrobacterium-mediated method.After plant selection and regeneration,a total of 52 and 29regenerated T1 and T2plants with resistance were obtained respectively.The PCR detection showed that a total of 41T1 plants and 23T2 plants containing LbMYB103 gene,and the real-time PCR analysis proved that LbMYB103 gene in transgenic A.thaliana could conduct normal expression.The phenotypic observation showed abnormalanther and flower development,shorten pods without seed both in T1 and T2transgenic A.thaliana,further indicating LbMYB103 gene might be related to the plant fertility.The results lay a foundation for further study on the LbMYB103 gene,which may play a regulating role in the process of wolfberry anther development.
出处
《西北植物学报》
CAS
CSCD
北大核心
2015年第9期1722-1727,共6页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金(31360361,31360025,31260065)
2012年宁夏高等学校科学研究项目
宁夏自然科学基金(NZ13031)
关键词
枸杞
LbMYB103
植物过量表达载体
拟南芥
遗传转化
wolfberry（Lycium barbarum L.）
LbMYB103
plant over-expression vector
Arabidopsis thaliana
genetic transformation
