Molecular Mechanisms Of The Dwarf Characteristic In Pinus Bungeana Zucc.ex Endl.

As a unique tree species in China,Pinus bungeana Zucc.Ex Endl.has important cultural value,ecological value and economic value.To enrich the molecular data of Pinus bungeana Zucc.Ex Endl.and analyze the factor of the dwarf mutant character,de novo assembly of transcriptome annotation was performed for two varieties of Pinus bungeana Zucc.ex Endl.that are obviously different in morphology.In addition,the Pinus bungeana transcriptome library was established.Among the library,the differentially expressed genes were screened,resulting that five unigenes were related to the morphology of the Pinus bungeana.And,the five genes were verified by RT-q PCR.Furthermore,the metabolites and the different matebolites were analyzed by using the Metabolomics.On the basis of the above,the levels of hormones in the two varieties were determined.Also,the c DNA of the pivotal genes were cloned by RACE and analysed by bioinformatics,including the Pb CKX1,PHP1,PRR1.The main results are as the followings:(1)Transcriptome analysis was carried out on the two varieties with obvious difference in tree morphology,and a total of 88 092 unigenes with an average length of 758 bp were obtained.In addition,through BLAST,42 667 unigenes(48.43%)were similar to known protein databases.The difference analysis showed that there were a total of 98 up-regulated unigenes and 60 down-regulated unigenes,and there were different genes related to plant hormone transduction,cytokinin oxidase and other functions,including CKX,ARR-A,AHP and MADS-box transcription factor genes,which were closely related to the apical dominance and caused the difference in tree morphology.RT-q PCR results were consistent with the relative trend of transcriptome sequencing.(2)Using the Metabolomics to study the metabolic differences of Pinus bungeana with different plant morphology,a total of 3 864 annotated metabolites were obtained under the mode of positive and negative ions,among which 595 were differentiated metabolites.Among them,the metabolism of cytokinins such as N6-isopentenyl adenine and ZT were up-regulated.The tryptamine which is closely related to IAA was up-regulation.But,there are many pathways of auxin synthesis,therefore,cytokinins should be the main reason for the difference in tree morphology.(3)The change of condition of endogenous hormones between two different varieties of Pinus bungeana was found by using the LC/MS method.Relative to the ordinary varieties,plant hormone ACC and SA declined and the IAA,ZT and ABA increased.But only ZT changes extremely significant difference in statistics.And the ratio of IAA and ABA,ZT and IAA increased.The new variety's short stature and numerous lateral branches were probably closely related to the significant increase of ZT,the decline of IAA/ABA and the increase of ZT/IAA.(4)The full-length c DNA of the pivotal genes related to the loss of apical dominance in the Pinus bungeana variety were cloned and analyzed,including Pb CKX1,PHP1 and PRR1.The Pb CKX1 is related to the biosynthesis of CKX.The PHP1 and PRR1 are both related to the hormone signaling of CK.Bioinformatics analysis showed that the amino acid sequence encoded by Pb CKX1 was similar to that encoded by At CKX7 gene in Arabidopsis thaliana.The amino acid sequence encoded by PHP1 was similar to the AHP1 protein in Arabidopsis thaliana.The amino acid sequence encoded by PRR1 had the highest similarity with ARR8 and ARR9 proteins of Arabidopsis thaliana.Above all,we suggest that,the increase of CK was the main reason for the dwarf characteristic of Pinus bungeana,and the increase of IAA,ABA and the proportion of various hormones also played a certain role.The reason for the increase of CK was the down-regulation of the Pb CKX1 gene which encoded the CKX enzyme in Pinus bungeana.In addition,the increase of CK also caused the up-regulated expression of the genes related to the hormone signaling of CK.