Study On The Development Of Microtubers And Differential Display Of MRNA In Pinellia Ternata In Vitro

Pinellia ternata(Thunb.) Briet.enriched with the medicinal ingredients of protein, tannim and alkaloid is an important steroid medicine resources plant that is endemic to China.The degeneration in quality of Pinellia ternata,caused by virus diseases usually results in a serious loss of both tuber yield and quality,which has been a main hinder in production of PinelIia ternata,for long time.Virus-elimination through meristem culture has been widely used in the seedling and microtubers of Pinellia ternata.and the development of biotechnology is important channels to resolve the production of artificial seeds and the improvment yield and quality.The microtuber of Pinellia ternata.,which is induced from the upper of leaf stalks ends,has the same genetic stability,morphological and physiological characteristics compared with conventional tubers.Perspectives of using microtubers of Pinellia ternata,in seed system has,therefore,drawn great interests in research.However,the productivity of microtubers is relatively low since there is little information about the tuberization mechanism.With aims to look into this aspect,the experiments were laid out under various tuber inducing conditions and investigations of variation in endogenous hormones and expression of tuber-forming-relative-genes (TFRG) were carried out.The main results were as follows:Studied the effects of the inhibitor of gibberellins-biosynthesis on tuber enlargement and content of endogenous hormones of Pinellia ternata.Pinellia ternata microtubers were induced in the medium MS added with different concentration of CCC.The results showed that CCC could significantly add microtuber weight and inhibit the synthesis of GA₃,IAA,ZR and they were reduced as the content of CCC increased.Meanwhile,CCC could effectively influence the content of ABA and JA shaped as "V" and "J".Studied the effects of the inhibitor of abscissas acid -biosynthesis on tuber enlargement and content of endogenous hormones of Pinellia ternata.Microtubers were induced in the medium MS added with different concentration of NOR.The results showed that NOR could significantly reduce microtuber weight and inhibit the synthesis of ABA,IAA,JA and the content of JA shaped as"N".Meanwhile,NOR could effectively influence the content of GA₃ and ZR.Studied the effects of the inhibitor of jasmines acid-biosynthesis on tuber enlargement and content of endogenous hormones of Pinellia ternata.Microtubers of Pinellia ternata were induced in the medium MS added with different concentration of SHAM.The results showed that SHAM could significantly postponed the formation of microtuber time and promote the synthesis of GA₃,IAA,ZR,but the content of them were lower than collation.SHAM could effectively inhibited the synthesis of JA and decreased the content of ABA.A highly efficient method of isolating total RNA from microtubers of Pinellia ternata in vitro was developed in this study.By modifying the method recommended by Guanidinium for extracting total RNA from plant tissues rich in phenolic and polysaccharidic compounds,a simple and convenient method for extraction of total RNA from plant materials containing abundant polyphenols and polysaccharides was established.The value of RNA extracted with improved method A260/ A230 were all over 2.0 and the values of A260/ A280 were between 1.7 and 2.0.The electrophoresis bands cleared on agarosegel and integrity of RNA was good.The results showed that RNA obtained from rapeseed with this method had high purity and quality and could be used in molecular biological research,as DDRT-PCR and analysis directly.Establishment and optimization of sliver staining Differential Display of Pinellia ternate microtubers in vitro.The effects of five kinds of factors were studied by orthogonal design method including emplate cDNA,Mg²⁺,dNTPs,primers and Taq DNA polymerase,and in order to establish the optimum DDRT-PCR system of P.ternata. microtubers in vitro.A satisfactory DDRT-PCR technique system for P.ternata. Microtubers in vitro with desirable repeatability and polymorphic bands was established. In a total volume of 20μL DDRT-PCR system,it contained 10×buffer,150μmol/L dNTPs,2μmol/L anchor primer,1μmol/L arbitrary primer,2.5 mmol/L Mg²⁺,0.6 U Taq DNA polymerase and 2.5μg template cDNA.The effect of the five factors was in sequence of Taq DNA polymerase＞template cDNA＞dNTPs＞Mg²⁺＞Primers.Identification of expressed gene fragments related to the microtubers develop- ment of Pinellia ternat in vitro,by DDRT-PCR.In this study,we identified and characterized eight cDNA fragments differentially expressed in the induction of microtubers of Pinellia ternata in vitro,using mRNA differential display DDRT-PCR.Deduced amino -acid sequences of five of the eight cDNA fragments showed no significant homology with ESTs and genes in the Genbank databases.However,the remaining three showed significant homologies with sequences encoding components of eukaryotic translation initiation factor 3 subunit H,MADS-box protein and ethylene signal transcription factor, respectively.Their differential expression patterns were confirmed by semi-quantitative RT-PCR analysis.And the expression level of the induction of microtubers of Pinellia ternata in vitro.were different.The three cDNA fragments deduced like protein were most likely involved in the microtubers development of Pinellia ternata,and the study gave us novel insights into the molecular mechanism of the microtubers development of Pinellia ternata.