| Rehmannia glutinosa Libosch. is a large quantity of Chinese herb, whichis famous and used frequently in China. Because of its efficacy in regulating immune,anti-tumor, anti-aging and hpyerglycemic, etc. it is widely used in medical treatment,health care, food and other fields and its demand is becoming more and more largerin recent years. However, the land must be given up after one season of Rehmanniaglutinosa cultivation or replanted after a period of8-10years because of consecutivemonoculture problem which severely restricts the sustainable development ofRehmannia glutinosa. Therefor it has become an urgent problem to relieve andeliminate the consecutive monoculture problem of Rehmannia glutinosa.For the complex causes which form consecutive monoculture problem of Rehma-nnia glutinosa, people have a wide range of research and discussing on the problem,however, the molecular mechanism which causes continuous cropping obstacles isstill not clear. In order to discuss the mechanism and reduction technology ofconsecutive monoculture problem effect in Rehmannia glutinosa, we coustucted thesuppression subtractive library of continuous cropping Rehmannia glutinosa for thefirst time and did the analysis of Bio-Informatics using R. glutinosa cultivar "Wen85-5" as an experimental material in our study, From that, we screened somedifferential expression genes and did the spatio-temporal expression and functionalanalysis of these genes. Main research results are as follows:1. Construction of the cDNA subtructive libararies of Rehmannia glutinosa: totalRNA (the first year planting and the second year planting) were extractedrespectively. Single-stranded and double-stranded cDNA were synthesized andfurther digested using Rsa I, so as to get the Driver cDNA and Tester cDNArespectively. Tester cDNA were divided into two portions which were ligatedseparately with two different kinds of cDNA adaptor. The ligated cDNAswerehybridized twice and PCR amplified twice. The secondary PCR products wereinserted into pMD-18T vector and transformed into E.coli DH5α competentcell.Identification of the inserted cDNA fragments in subtractive library was done using PCR. More than1000positive clones were obtained. The results showed thatthere were inserted fragments of250-750bp in selected positive clones. The resultwas that got the high efficient forward and reverse cDNA libraries.2. Sequencing result and bioinformatics analysis: Selecting randomly300positive clones from forward and reverse cDNA libraries, respectively. The forwardand reverse libraries got different ESTs, and produced241(forward library) and232(reverse library) unique ESTs by sequencing.9unique sequences were repeatedbetween the two libraries. Through fuctional analysis in NCBI database,232and214differential ESTs were obtained from forward and reverse libraries, and there were200genes with function annotation in forward library and195genes in reverselibrary.According to the accession in COG database, genes with function annotation orputative functionannotation were assigned to categories. Forward library genes wereassigned to16categories and Forward library genes were assigned to15categories.These genes mainly were related to energy production and conversion, amino acidtransport and metabolism, nucleotide transport and metabolism, carbohydratetransport and metabolism, coenzyme transport and metabolism, lipid transport andmetabolism, translation, ribosomal structure and biogenesis, transcription,replication, recombination and repair, cell wall/membrane/envelope biogenesis,posttranslational modification, protein turnover, chaperones, inorganic ions transportand metabolism, signal transduction mechanisms, intracellular trafficking，nuclearstructure, cytoskeleton, general function prediction only and function unknown.3. Spatiotemporal expression and significant analysis: Do the Spatiotemporalexpression and Significant analysis of the five genes in forward library (separatelycoding calcium-dependent protein kinase, s-adenosyl-methionine synthetase,Aminocyclopropane-1-carboxylate oxidase, methyltransferase, calpain) and the otherfive genes chosen from the reverse library (separately coding RNA-dependent RNApolymerase, RNA replicase, DNA-directed RNA polymerase IIa, cyclin D, RNAbinding protein), respectively. The results showed the five genes chosen fromforward library had high expression in consecutive monoculture R. glutinosa,especially in root, while were hardly expression in normal growth R. glutinosa.Furthermore, the five genes had significant difference nearly in every stage and thebiggest difference appeared in expanding period of root. On the contrary, the otherfive genes chosen from the reverse library had high expression in one-year R.glutinosa, but were down regulated or no expression in consecutive monoculture R.glutinosa. And significant difference appeared in every stage and the biggestdifference occurred in elongating and expanding stage of root. On the one hand, theresults indicated that it is successful to construct the forward and reverse liabrary, Onthe other hand, it also indicated the10genes may play the important roles in thecauses which formed consecutive monoculture problem of R. glutinosa.4. Related function analysis of the10genes: Calcium-dependent proteinkinase and calpain mainly participated in calcium signal transmission andprotein decomposed. s-adenosyl-methionine synthetase and Aminocyclopropane-1-carboxylate oxidase mainly participated in ethylene synthesis. methyltransfer-ase mainly participated in physiological regulation and dye modification. the5above mentioned genes from forward library were all highly expressed inconsecutive monoculture R.glutinosa. RNA-dependent RNA polymerase, RNAreplicase and DNA-directed RNA polymerase IIa mainly participated intranscription regulation. cyclin D mainly participated in cell growth anddivision. RNA binding protein mainly participated in gene expression regulationand stress respone.The5genes which were chosen from reverse library wereall down regulated or shut off in consecutive monoculture R. glutinosa.Combining suppression subtractive hybridization with quantitative PCR, theresearch constructed the subtractive libraries of R. glutinosa for the first time, andobtained a lot of EST sequences which were related with consecutive monocultureproblem of R. glutinosa. What’s more, the research revealed the expression traits ofthe genes which responded to the consecutive monoculture problem of R. glutinosa,preliminarily. And that laid the foundation for the research on molecular mechanismsof consecutive monoculture problem of R. glutinosa. Through function analysis ofthe10key genes, the research described primarily the “poison” mechanism ofconsective monoculture R. glutinosa, which laid the theoretical foundation forfurther research the causes of consecutive monoculture problem, unscrambling themolecular mechanisms of consecutive monoculture problem and developing theeffective technology of alleviating the harm of consecutive monoculture. |
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