Isolation And Expression Of Genes Related To The Flower Development From Tea (Camellia Sinensis) Plant

Tea plant(Camellia sinensis)is a kind of important economic crop in China. There have more than 8000 thousand farmers to cultivate tea plant,and the production value of tea is average 37.5 million dollars every year in China.It begins to flower and fruit after being planted 2-3 years.Under nature pollination condition,the flowering rate of most tea plant varieties usually is very high.The flower bud differentiation of tea plant usually starts in June,lasts to December or even later.It takes about six months from bud differentiation to fruit.The tea plant needs to be consumed lots of energy and nutrients to fill for its reproductive growth,however,the vegetative growth especially the tender leaves growth is restrained.As a result, reproductive growth is always reducing the product of tea leaves and declining the quality of tea plant,which directly decreased the income of tea farmers.Accordingly, it has been an important issue to control the reproductive growth for the need of either the quantity increasing or the quality promoting of tea plant.In general,tea plant reproduction uses of cutting propagation in order to stabilize good character in heredity.Separation of the generations of tea plant is unable to use the same genotype F1 by crossing fertility.But in the cutting propagation process, nursery stock requires specially to care in transport with equipment and material.In addition,there have many disadvantageous factors,which pests or diseases spread easily,as well as resistance weaker than seedlings compared with seed reproduction. Under nature pollination condition,the fruiting rate is about 2%by self-fertilization, and heterosis of character of tea plant filial generation is unavoidable due to the limitation of its self-fertility.Heterosis making use of the male sterile breeding be able to save large amount of emasculation labour,ensuring that seed purity.It is possible that breeding can be solved while the trend of single tea varieties and genetic breeding the problem of insufficient resources.At present,the research of specific characteristic product related with the quality of tea is developing fast,but there is little research on the flowering period of tea plant in molecular biology.Therefore,it elucidate the molecular mechanism of tea flower development for studying on the expression of genes related to male fertility of tea plant,and it also lay the theoretical foundation for controlling male fertility to enhance the production of tea.Here,we chose two kinds of significant differences in the rate of fruiting of Longjing 43 and Wulong,and focus on the following research: One is of the gene differential expression in tea flower bud at different development stage.The cDNA-amplified fragment length polymorphism approach was used to identify genes expressed differentially during early and late flower bud development in tea plant;Another is of the cloning,analysis the genes related to male fertility through fragments from derived transcript by cDNA-AFLP.The results are as following:1.To optimize the datum,the results showed:preamplification more suitable annealing temperature of 53℃,20 amplification cycles for the cycle preamplification and selective PCR amplification system 2.3mM Mg²⁺concentration by using cDNA-AFLP.2.For studies of differential gene expression,cDNA-AFLP was analyzed in tea plant at the stage of bud flower development.The result showed that there are evident differential expressions in flower between the early and late stages.Of the 1110 fragments inspected,87 specially displayed in tea flower bud at the late stage;15 fragments were cloned and sequenced.Sequence analysis indicated that among 15 squenced fragments,nine were sequence-similar to proteins in GenBank,i.e. oxidoreductase,chromosome 13,14-3-3 protein,Amyrel gene,calreticulin interacted protein,ATP sulfurylase,catalytic/iron ion binding,desiccation-related protein and ntp302,two were similar to hypothetical proteins,and no similar sequence were found in GenBank for the other 4 fragment.3.The complete cDNA sequence of 14-3-3 gene was isolated from flower buds at the late stage by Rapid amplification of cDNA ends(RACE).The full-length cDNA of 14-3-3 sequence is 1072bp(GenBank accession number DQ444463)and contains a 780-nueleotides-long open reading frame starting at position 68 and terminated by a stop codon(TAA)at position 848-850,and a 193-nueleotide-long 3'-untranslated region plus a 29-monomers-long poly(A)-tail.The encoded protein of 260 amino acids in length had a predicted molecular mass of 29.4 kDa and an isoelectric point (pI)of 4.73.Homology searches with the dudueed 260 amino acid residues indicated that 14-3-3 of tea(Camellia sinensis)had a high identity with other plant 14-3-3 proteins,especially that of the hybrid poplar(Populus x canescens,86%identity, accession number AAD27823),by reason of both of them belonging to the arbor.This open reading frame with 0.78kb was cloned into the high-expression vector PET32a,under control of the T7 promoter.Expression of this plasmid in Escherichia coli BL21 resulted in the production of a fusion protein(≈49kDa)consisting of 112 amino acids of thioredoxin and 260 amino acids of 14-3-3 gene.The plasimid of PET32a contain 14-3-3 ORF in Escherichia coli BL21 was the highest expressional products(34.6%)after IPTG induced for 3h.Antigen preparation was separated from SDS-PAGE and was purified by running gradient SDS-PAGE.To immunize the mice with the pure 14-3-3 protein produced immune sera.Immunobolts analysis revealed some of mice produced the 14-3-3 antibody in immune sera.Immunoblots showed the presence of 14-3-3 protein in the flower buds at the late stage,but absence of it in tea flower buds at the early stage, root,stem,and leaf,and 14-3-3 gene of differential expression patters was assessed by RT-PCR.The presence of 14-3-3 protein and Fusicoccin can improve pollen germination and pollen tube length of tea flower bud in vitro.It was suggested thatl4-3-3 gene encodes protein maybe involve in cell signaling transduction and trafficking events during the late stage of flower bud development.Furthermore,14-3-3 protein was associated with the germination of pollen grains and the growth rate of pollen tubes.4.A transcript derived fragment,TDF45,was obtained via selective amplification with E12/M24 primer pair.TDF45 was specifically expressed only in the flower buds at the late stage by RT-PCR.The complete cDNA was recovered by rapid amplification of cDNA ends,then cloned and sequenced.It was found that the full-length cDNA of 1736bp(GenBank accession number DQ444464)included a 1404bp open reading frame predicting a 467-amino-acid polypeptide of 52.2 kDa. Analysis of the nucleotide sequence and deduced amino acid sequence revealed a high homology with pollen specific proteins from patato and Arabidopsis.Thus,we propose that this cDNA encodes the first ATP sulfurylase described in tea plant, designated APS.5.A transcript derived fragment,TDF53,was obtained via selective amplification with E12/M20 primer pair.TDF53 was specifically expressed only in the flower buds at the late stage by RT-PCR.The complete cDNA was recovered by rapid amplification of cDNA ends,then cloned and sequenced.It was found that the full-length cDNA of 2079bp(GenBank accession number DQ887753)included a 1701bp open reading frame predicting a 567-amino-acid polypeptide of 63 kDa. Analysis of the nucleotide sequence and deduced amino acid sequence revealed a high homology with pollen specific proteins from tobacco and Brassica napus.Thus,we propose that this cDNA encodes the first pollen specific protein described in tea plant, designated CsPSP.