Study On Genetic Diversity And Differentially Expression Of Genes Induced By Cold-Stress In Acacia Melanoxylon

Acacia Melanoxylon is a Mimosaceae Acacia Mill, naturally distributed from south Brisbane, Australia to the south-east of South Australia and Tasmania. It is one of the main timber tree species in South China, which was imported within recent years. It features easy adaptation, high yield and broad range of usages and so forth. However, because of original from tropical regions, the most of them are still very sensitive to low temperature. Real practice has proved that Acacia Melanoxylon imported into the north Fujian & the further northern regions grows poorly. Low temperature is the main limit in wintertime. In addition, it's seedling traits in cultivation with significant differentiation, restricting plantation development of Acacia Melanoxylon of the high-yield and high-quality. Therefore, the study of Acacia melanoxylon plus tree, genetic diversity, cold tolerance and identification of biological function of genes expressed differentially under cold stress can provide a theoretical basis for further clarification of the cold-resistance mechanism of Acacia melanoxylon and cultivating good character and stability of the cold Acacia strains. In this study, ISSR molecular marker has been used to analysis of genetic diversity in Acacia melanoxylon plus tree, cold-assessment techniques have been used to test cold tolerance of Acacia melanoxylon, cDNA-AFLP technology has been used to to analysis differentially expressed genes in cold-induced .The main results are as followings:1 A fast and convenient method for extraction of DNA from Acacia Melanoxylon is established.1.5×CTAB method is considered as one of fast and convenient methods for extraction of DNA in young fresh leaves of Acacia Melanoxylon . The results indicates that 180-697.5μg·g-1DNA are obtained from a phyllode , their molecular weights are about 48 Kb, and the A260/A280 of DNA are 1.75～1.955,the DNA is suitable for digesting with restriction enzyme and ISSR reaction without purifying by RNase.2 ISSR-PCR system of Acacia Melanoxylon has been established and optimized with uniform design, screening ten of ISSR primers with rich polymorphism and good stability, optimizing ISSR amplification procedures and primer annealing temperature of Acacia Melanoxylon.(1) ISSR-PCR system of Acacia Melanoxylon: Each 20μL ISSR-PCR amplification reaction solution is consisted of 3.00 mmol·L-1 Mg2+ , 0.15 mmol·L-1 dNTP, 0.30μmol·L-1 Primer , 0.75U Taq DNA polymerase , 2.00 ng·μL-1DNA template.(2) ISSR amplification procedures of Acacia Melanoxylon: The suitable ISSR-PCR procedure was 1 cycle of pre-denaturing for 5 min at 94℃, 33 cycles of denaturing for 35s at 94℃, annealing for 52s at 52-61.5℃, extending for 90s at 72℃and the last cycle of extending for 10 min at 72℃.3 Verification to Genetic diversity of Acacia Melanoxylon based on ISSR markersGenetic diversity among 37 Acacia Melanoxylon plus trees is detected by ISSR markers. The results reveal a high level of genetic diversity among 37 Acacia Melanoxylon plus trees, having a low level of genetic diversity between different Acacia Melanoxylon plus trees that are planted in different region in Fujian. This results are matching background information of Acacia melanoxylon and cultivation facts. The Nei & Li(1979) genetic distance among 37 Acacia Melanoxylon plus trees reveals a high genetic differentiation among 37 Acacia Melanoxylon plus trees. Relatively, 3 plus trees from Jianou, Fujian have a high level of genetic diversity; 16 plus trees of good clones from Guangdong have a high level of genetic diversity with a low genetic differentiation. 7 seedlings of good clones from Guangdong have the lowest level of genetic diversity. The results show that: ISSR molecular marker technique is suitable to be applied to analysis of the genetic diversity of Acacia Melanoxylon .Cluster Analysis show that: genetic relationship between 3 plus trees from Jianou, Fujian and the other 34 Acacia Melanoxylon plus trees maybe is far. 16 plus trees of good clones from Guangdong cluster together with half-sib 7 seedlings of good clones, which reveals that they are genetically related. This is matching background information of Acacia melanoxylon and cultivation facts. Study shows that: ISSR molecular marker technique is suitable to be used for identification of genetic relationship of Acacia Melanoxylon .In addition, 10 primers for amplification of three cold-resistance plus tree are amplified in 48 specific genetic loci, in which a total of three cold-resistance plus tree-specific genetic loci, 22, can be used as difference from other 34 plus tree of ISSR molecular markers; Another 26-specific genetic loci with a total of 22 specific genetic loci used in conjunction, can be used as ISSR molecular markers to distinguish between three cold-plus tree.4 Cold-resistance assessments on Acacia melanoxylonThe relative electrical conductivity of Acacia Melanoxylon under low- temperature treatment was measured by electrical conductivity method. The turning point temperature (semi lethal temperature) measured by Logistic curve is used as the index of cold-tolerance, may directly and effectively show cold resistance and low temperature limit of Acacia melanoxylon, so this method is an effective one for cold-resistance assessment on Acacia melanoxylon.Cold resistance of Acacia melanoxylon is associated with the cumulative rate of MDA content of cells under low-temperature treatment. MDA content of cells accumulated a small extent in cold tolerant clones. Meanwhile, MDA content of cells is accumulated a large extent in weak cold resistance clones. Results show that cumulative rate of MDA content of cells can be used as detective index of cold-resistance assessment on Acacia melanoxylon.The relationship between soluble sugar content and cold resistance is complicated. Some provenances of Acacia melanoxylon, soluble sugar content are negatively correlated with cold resistance, revealing indirect effect of soluble sugar accumulation in the cold resistance .The relationship between proline content and cold resistance is complicated. Some provenances of Acacia melanoxylon, proline content are negatively correlated with cold resistance. Increasing of proline content leads to increase osmotic, inducing other processes of cold resistance or result low-temperature stress only.5 A fast method suited to extract total RNA applied to cDNA-AFLP from plantlets of Acacia melanoxylon cultured in vitroThe modified CTAB- guanidinium isothiocyanate method can efficiently remove polyphenol, polysaccharide, proteins and DNA that often contaminate with RNA. The high-quality and integrity total RNA acquired the 28S rRNA, which is approximately two times as light as the 18S rRNA with 1.96-2.0 of OD260/OD280 ratio and 1.98-2.2 of OD260/OD230 ratio, and the total RNA yield of 118.4-213.6μg.g-1, and the quality of extracted RNA meet the requirement of subsequent molecular biology reactions as in dscDNA synthesis and cDNA-AFLP6 Establishment of cDNA-AFLP analysis system with two low- frequency cut Enzyme in Acacia MelanoxylonAn optimized system is established after studying on the factors affecting cDNA-AFLP analysis system of Acacia Melanoxylon cultured in vitro. The results indicates that the restriction enzyme combination is EcoR I and PstⅠ,the dosage of dscDNA is 500 ng ,the ligation products used as template of pre-amplification are diluted to one time ,and the pre-amplification products used as template of select-amplification are diluted to50 times. 7 Isolation and identification of differentially expressed genes cold -induced in Acacia melanoxylonThe transcription profile of Acacia Melanoxylon response to cold stress is analyzed by cDNA-AFLP, and 147 differentially expressed TDFs (transcript-derived fragments) is sequenced functional annotated by Blast in NCBI. And the TDFs are further classified into 2 subgroups:Ⅰup-regulated expression (140)Ⅱdown-regulated expression (7). There are zinc finger protein, GTPase activating protein, guanine nucleotide exchange factor, protein phsophatase-2A, myb domain-containing protein, transcription factor NAC, receptor-like protein kinase (RL Ks), protein tyrosine phosphatase,receptor B(PTPRB), Ribonucleoside-diphosphate reductase, ribulose-1,5-bisphosphate carboxylase, cytochrome P450, carboxylic ester hydrolase, urease accessory protein ureG, H+-ATPase, ribosomal protein S7, Cyclin-related protein, Thioredoxin(Trx).The results of this study will build up the foundation for study on the mechanism of cold resistance and the possible role of responsing to low-temperature of TDFs corresponding to differentially expressed genes in Acacia melanoxylon.