Study On The Substances Growth And The Expression Of Accase Encoding Gene From Aleurite Moluccana

Aleurites moluccana belongs to woody-oil plants in Euphorbiaceae. Because of its high oil content in seeds. It was a potential bio-energy species.The main purpose on this test was to clear the relationship between the substance changes and ACCase encoding gene from wild Aleurites moluccana.First of all,studying on the changes of fruit size and substances in different stage from Aleurites moluccana. Second,the fruits were used as materials,then the housekeeping gene 18S rRNA was cloned by RT-PCR technology.In the end,Using fluorescence quantitative real-time PCR technology to study on the expression of ACCaseencoding genein fruits at different stages from Aleurites moluccana which would provide a research method between fat content and ACCase encoding gene in the future.1.The change of fruit size and substance from Aleurites moluccanaThree wild Aleurites moluccana plants at different flowering stage (75d、 105d、135d、165d、195d) were randomly selected as material.The changes of fruit size and content of soluble protein,sugar,starch,fat and water were mainly studied in Aleurites moluccana.The results showed that:the vertical diameter.transverse diameter and side diameter of fruit exhibited"S"shape curve during fruit development,and the three diameters changed a little after 75 days of flowering.They were reach to 81.99%、85.00%、86.94% compared to the mature fruits.The fruits were completely mature after 75 days of flowering.Combining three plant the changes law of soluble protein was basicly similar to the soluble sugar.Both of themstarted raise up after 75 days of flowering,after 135 days of flowering they both reached to the maximum which the content was 41.92mg/g and 23.29mg/g respectively.Then it began to decrease after 195 days of flowering.The changes of soluble starch reached to the maximum after 105 days of flowering(13.87mg/g).Then it decreased rapidly.After the increasing of water content in 105 days of flowering,it performanced to continue decreasing until mature.The fat content showed to constantly accumulate.The maximum of fat content was 67.54% after 195 days of flowering.2.Cloning and sequence analysis of 18S rRNA gene fragment from Aleurites moluccanaFirstly, according to the 18S rRNA acid sequence of Jatropha curcas(accession No.AY823528.1)、Arabidopsis thaliana(accession No.GQ380689.1)、Camellia japonica(accession No.U42815.1)、Ricinus communis(accession No.AB233559.1) and Glycinemax(accession No.X02623.1),using Primer 5.0 to design the specific primers.Then the fruit was used as material and RT-PCR technology was used to clone the housekeeping 18S rRNA gene sequence.We obtianed a 110bp nucleotide.The results of GeneBank showed that the degreeof identity of 18S rRNA nucleotide sequence between 18S rRNA gene of Aleurites moluccana and that of known homologous plants was 100%.This indicated that the cloned fragment was 18S rRNA gene fragment.3 The expression analysis of ACCase encoding gene18S rRNA gene was made as reference gene.To explore the relationship between ACCase encoding gene and fat accumulation.we studied on the expression of ACCase encoding gene ACCB、ACCC、ACCD in fruits from wild Aleurites moluccana plants at different stages. The result showed that:The amplification efficiencies of 18srRNA gene、ACCB gene、ACCC gene、ACCD gene was 91%、107%、104% and 91%.The linear correlation coefficient was 0.997、0.998、0.996 and 0.996. The expression of three ACCase encoding gene was significantly difference in different Aleurites moluccana plants. The transcription level of ACCB gene was highest after 135 days of flowering.However the highest expression of ACCC and ACCD gene was after 165 days of flowering.135 days and 165days of flowering were not only the most active period of these three encoding gene but also the fastest period of fat accumulation.This indicated that the highest transcription level of these three genes had a close relationship with the fat accumulation.