【作者】 王玉红；

【导师】 陈新建；

【作者基本信息】 河南农业大学 ， 生物化学与分子生物学， 2019， 硕士

【摘要】 由于近年来,中医兴起,人们对中药的需求也与日俱增。地黄(Rehmannia glutinosaL.)作为我国重要的大宗药材之一,远销海内外,但其栽培与种植却存在严重的连作障碍,极大的影响地黄的产量和品质,探寻其分子机制已成为地黄研究领域的重要问题之一。基于前期RNA-Seq结果,筛选到特异响应连作障碍的转录本Unigene29334_All,利用hiTAIL-PCR并结合RACE技术克隆获得长度为1573bp的完整转录本。通过ORF Finder分析发现,所克隆片段全部ORF均小于300nt,表明此转录本为长链非编码RNA(Long non-coding RNA,LncRNA)。结合与地黄转录组数据库的比对发现,此LncRNA最大ORF与丙氨酸乙醛酸转氨酶2编码基因(Alanine--glyoxylate aminotransferase 2,AGT2)部分区域同源,因此,将克隆获得的LncRNA命名为LncRNA-RgATG2。为进一步探究LncRNA-RgAGT2的功能,本研究检测了LncRNA-RgAGT2与RgAGT2基因在头茬和连作的5个地黄生长发育关键时期(苗期,拉线期,膨大前期,膨大中期,膨大后期)的表达模式,其结果暗示了,LncRNA-RgAGT2可能与RgAGT2基因存在调控关系。同时,LncRNA-RgAGT2在连作地黄各个时期均呈现显著波动,可以作为“标记基因”对连作地黄进行早期诊断。本研究首次发现LncRNA参与地黄连作障碍应答及调控过程,是对地黄连作障碍分子机制的有力补充。为了进一步研究LncRNA-RgATG2在地黄连作障碍中的作用,本研究构建了LncRNA-RgATG2RNAi沉默载体,通过农杆菌介导的叶盘转化法获得LncRNA-RgATG2沉默的阳性植株。结果发现,部分LncRNA-RgA TG2沉默植株在无激素处理的正常生根培养基上出现膨大现象,且LncRNA-RgAGT2与RgAGT2基因表达均呈现下调,进一步证实了LncRNA-RgAGT2与RgAGT2基因通过某种途径共同参与地黄块根膨大调控。基于前期课题组通过调节激素浓度诱导地黄在MS培养基生根膨大,本课题利用激素(6-BA和NAA)对LncRNA-RgATG2沉默的阳性植株进行处理诱导膨大,观察激素对LncRNA-RgATG2沉默植株块根膨大的影响,结果发现,在激素处理下未经侵染的再生苗和LncRNA-RgATG2沉默植株均出现膨大,且LncRNA-RgAGT2与RgAGT2基因均出现下调,但是LncRNA-RgATG2沉默植株块根明显比再生苗膨大,同时,与无激素处理的正常生根培养基上膨大的LncRNA-RgATG2沉默植株相比,激素处理加速了 LncRNA-RgATG2沉默植株的膨大。由此推测LncRNA-RgATG2参与地黄块根膨大与激素有关,可能与RgAGT2共同通过激素信号应答途径来参与地黄块根膨大调控。本课题通过对响应地黄连作障碍的LncRA-RgATG2功能的初步分析,来探索LncRNA-RgATG2在地黄连作障碍中的作用,从而进一步揭示地黄连作障碍的分子机制。更多还原

【Abstract】 In recent years,the demand for Chinese medicine has increased because of the rise of traditional Chinese medicine.Rehmannia glutinosa L.(R.glutinosa)is one of the most important medicinal materials in China which is exported to home and abroad.However,in its cultivation and planting,there are serious continuous cropping obstacles,which greatly affects the yield and quality of Rehmannia glutinosa.The search for its molecular mechanism is one of the important issues in the research field of R.glutinosa.This study was based on the previous response RNA-Seq screened for the unique response to the continuous disorder transcript Unigene29334_All,and cloned the complete transcript with a length of 1573 bp using hiTAIL-PCR and RACE techniques.The ORF Finder analysis showed that all the ORFs of the cloned products were less than 300nt,and the transcript was confirmed to be long non-coding RNA(LncRNA).In comparison with the R.glutinosa transcriptome database,this LncRNA is homologous to a portion of the gene coding for alanine glyoxylate transaminase 2(RgAGT2).Therefore,the LncRNA was cloned and named LncRNA-RgATG2.To further study the function of LncRNA-RgAGT2,we examined the expression patterns of LncRNA-RgAGT2 and RgAGT2 genes in the five critical periods(seedling stage,Drawing period,Expansion Prophase,Expansion medium,Later expansion period)of growth and development of the first year plant(FP)and the continuous cropping of R.glutinosa.The results suggest that LncRNA-RgAGT2 may have a regulatory relationship with RgAGT2 gene in R.glutinosa.At the same time,LncRNA-RgAGT2 exhibited significant fluctuations in all stages of R.glutinosa,and it can be used as a"marker gene" for the early diagnosis of continuous cropping yellow.For the first time,this study found that LncRNA participates in the response and regulation process of R.glutinosa,and it is a powerful complement to the molecular mechanism of rehabilitative stem disease in R.glutinosa.In order to further study the role of LncRNA-RgATG2 in the continuous cropping disorder of R.glutinosa,we constructed the RNAi silencing vector of LncRNA-RgATG2 and obtained the positive plants of LncRNA-RgATG2 silence by Agrobacterium-mediated leaf disc transformation.Based on the previous work of regulation of hormones concentration induced expansion of R.glutinosa roots cultured in MS medium,this work treated the positive plants of LncRNA-RgATG2 silence with(6-BA and NAA)to induce expansion of roots,and observe the effect of hormones on root enlargement of LncRNA-RgATG2 silent plants.The results showed that both the uninfected regeneration seedlings and the LncRNA-RgATG2 silenced plants were expanded under hormone treatment,and the LncRNA-RgAGT2 and RgAGT2 genes were down-regulated,but the roots of LncRNA-RgATG2 silenced plants were significantly larger than the regeneration seedlings.At the same time,hormone treatment accelerated the expansion of LncRNA-RgATG2 silenced plants compared to the expanded LncRNA-RgATG2 silenced plants on hormone-free normal rooting medium.It is speculated that LncRNA-RgATG2 is involved in the expansion of the roots of R.glutinosa and is related to hormones.It may be involved in the regulation of R.glutinosa roots enlargement through the hormonal signal response pathway together with RgAGT2.In this study,we explored the role of LncRNA-RgATG2 in the continuous resistance of R.glutinosa by analyzing the function of LncRNA-RgATG2 in response to the obstacles of R.glutinosa,thereby further revealing the molecular mechanism of the continuous resistance of R.glutinosa.更多还原