Study On Biochemical And Molecular Mechanisms Of Larix Leptolepis ×L. Principis-rupprechtii Somatic Embryogenesis

Somatic embryogenesis is one of the important large-scale asexual propagation methods for conifers, and has great application value in rapid propagation, conservation of germplasm, genetic transformation and other fields. Furthermore, it can be employed as an important model system for studying cell oftotipotency. It is very helpful that studying the mechanism of somatic embryogenisis, not only to solve the problems existing in somatic embryogenisis, but also to clarify the essential of the cell division approach of plant somatic embryogenesis. In this study, six development periods duiring somatic embryo maturation of embryogenic cell line Y35 of Larix leptolepis×L. principis-rupprechtii were demarcated according to morphological alterations, then using the embryogenic callus and the cultures of different development periods of Y35 as experimental mertials, the change trends of contents of soluble protein, soluble sugar and starch as well as the activity of SOD, POD and CAT were studied, and differentially expressed cDNA libraries for the stages of proembryogenic mass and somatic embryo maturation of Y35 were constructed by the technology SSH, then the expression profile of 25 genes belonging to 8 types were analysed using semi-quantitative RT-PCR and Real-Time PCR. The main results are as follows:1. The PEMⅢof Y35 underwent early single embryo, middle single embryo and late single embryo orderly, and finally developed to mature cotyledonary embryo at the stage of somatic embryo mature: the period of PEMⅢdeveloping to early single embryo was from the first day to the 5th day after embryogenic callus being induced, the period of early single embryo developing to middle single embryo was from the 5th to the 14th day, the period of middle single embryo developing to late single embryo was from the 14th to the 21th day, the period of late single embryo developing to early cotyledonary embryo was from the 21th to the 28th day, the period of cotyledonary embryo maturation was from the 28th to the 42th day, and among which the period of early cotyledonary embryo developing to middle cotyledonary embryo was from the 28th to the 35th day, the period of middle cotyledonary embryo developing to late cotyledonary embryo was from the 35th to the 42th day.2. The reseach on change trend of the contents of soluble protein, soluble sugar and starch showed, during the somatic embryogenesis of Y35, the soluble protein content was from 2.503 to 10.782 mg·g-1FW, showed an ascending trend and presented two accumulation fastigiumes respectively from PEMⅢto middle single embryo starting forming and cotyledonary embryo maturation. Soluble sugar content was from 6.662 to 10.543 mg·g-1 FW, and starch content from 5.566 to 14.970 mg·g-1FW, taking on an up-down-up trend, increasing in the periods of early single embryo developing to middle single embryo and cotyledonary embryo maturation, while decreasing during the middle single embryo developing to the cotyledonary embryo.These indicated that the early stage of somatic embryogenesis and the period of cotyledonary embryo maturation were the phases for energy accumulating, and the periods of cotyledonary embryo forming and somatic embryogenesis germination were the phases of energy consuming.3. The reseach on activity changes of SOD, POD and CAT during the somatic embryogenesis of Y35 showed:(1) The activty of SOD was from 52.98 to 133.20 U·g-1·h-1, and remained a rising trend after early single embryo forming, this revealed that SOD might be positively correlated to the differentiation of embryogenic cell and the development of somatic embryo.(2) The activty of POD was from 0.05 to 0.50 U·mg-1·min-1, ascended firstly and desceded later, and was highest in embryogenic callus and lowest in late single embryo , this revealed that POD might be positively correlated to the division and differentiation of proembryo mass, while negatively correlated to the development of PEMⅢto late single embryo.(3) The activty of CAT was from 0.86 to 2.81 U·mg-1·min-1, showed an up-down-up trend, reaching to the highest peak at the time of early embryo formating and decreasing to the lowest at the time of early cotyledonary embryo formating, this revealed that CAT might be positively correlated to the development of early single embryo, while negatively correlated to the formation of middle single embryo and early cotyledonary embryo.The changes in activty of SOD, POD and CAT indicated these three antioxidant enzymes coregulated the differentiation and development of embryogenic cells during Larix somatic embryogenesis.4. Differentially expressed cDNA libraries of the stages of proembryo mass and somatic embryo maturation were successfully constructed by suppression subtractive hybridization (SSH). 868 UniESTs were obtained, 400 of them gained from the subtractive library of stage of proembryo mass and 468 from that of somatic embryo maturation. 516 functionally known genes were obtained after Blastx analysis, 237 of them were from the subtractive library of stage of proembryo mass and 279 from that of somatic embryo maturation. 270 no hit found UniESTs were found, 111 of them from the subtractive library of stage of proembryo mass and 159 from that of somatic embryo maturation. 82 unclassed UniESTs were also obtained, which had low identity with its matched sequence or had high identity but its function was unknown, among them, 52 from the subtractive library of stage of proembryo mass and 30 from that of somatic embryo maturation.5. All the UniESTs could be classified into 19 categories according to their function. In the functionally known genes, the proportions of metabolism-related genes were highest, especially the carbohydrate metabolism-related genes; the next were the genes related to signal transduction, protein synthesis or degradation, transport facilitation and transcription; the genes related to cell structure, nuclear acid and protein processing, cell growth and division and storage protein also accounted for certain percents; while the genes related to development, cell fate, energy and defense occupied the lower percents. The percents of genes related to signal transduction, transcription, storage protein, cell fate and development all obviously increased in the stage of somatic embryo maturation, especially the those storage protein-related genes.6. The expression profiles analysis of 25 genes belonging to 8 types in different development periods of Y35 somatic embry showed, genes involved in transcription, signal transduction, cell fate, substance transport, cell and development regulation were active at early and middle stages of somatic embryogenesis, while at the stage of cotyledonary embryo maturation, the high level-expressed genes were mainly those related to storage protein, maturation and defense. These genes involved in different physiology and biochemistry processes all differentally expressed in the different development periods and presented same or different expression patterns, this revealed that they played important regulation roles at different periods during the somatic embryogenesis of Y35.These results showed complicated biochemical and molecular ragulation mechansim were involed in the process of Larix somatic embryogenesis, and somatic embryogensis was the result of the multifactors coregulating. This study not only lain a good foundation for the further application of the technology of somatic embryogensis in trees, but also provided important basis for solving the problems of life sciences.