Transcriptome Analysis Of Parakmeria Omeiensis Flower Buds And Effects Of B And C-Class Genes

Parakmeria omeiensis is a threatened plant belongs to the family Magnoliaceae and genus Parakmeria.The flowers categories into male and bisexual flowers in different plants.Male infertility in bisexual flowers,pistil is selectively aborted in male flowers after sex determination,so it is a precious material for research of flower organ morphogenesis and sexual differentiation caused by selective abortion.In addition,there have been controversies about the establishment of genus Parakmeria,and systematic studies are still being revised and improved.In this paper,we used transcriptome sequencing technology to screen out the differential genes related to sex differentiation,and cloned five B and C genes and analyzed for the effect.These results provide the basic support for the study of sex differentiation and its systematic evolution research of P.omeiensis.The main results are summarized as follows:1)Ten genes involved in hormonal signal transduction and genital development were selected from the transcriptome of the male and bisexual flower buds of P.omeiensis,including GA synthesis negative regulator GA2 ox,SAUR32 and IAA4 which encoding auxin response protein SAUR and AUX/IAA,COL1(involved in jasmonate signal transduction),EXL4(encoding pollen wall protein).In addition,30 MADS-box family genes with high expression levels were identified,and 5 B and C-class genes were found to be differentially expressed in male and bisexual flowers.2)7 MADS-box genes were cloned from P.omeiensis by homologous cloning,including 2 A genes,3 B genes,and 2 C genes,named Paom AP1,Paom AGL6,Paom AP3,Paom PI1,Paom PI2,Paom AG1,Paom AG2.Analysis of the C-terminal domain of the proteins encoded by these genes revealed that Paom AP1 has FUL motif and Paleo AP1 motif,Paom PI1 and Paom PI2 have PI motif,Paom AP3 has Paleo AP3 motif and PI-derived motif,and Paom AG1 and Paom AG2 have AGI motif and AGII motif.These elementary analysis showed that they are highly conservative,we speculated that the evolutionary position of P.omeiensis is primitive.The phylogenetic tree analysis showed that the subgenus Yulania were genetically distinct from the subgenus Magnolia.The Parakmeria is a solitary genus with close relationship with Michelia L.and Subgenus Yulania,but far from Magnolia subgenus.3)The q RT-PCR technique was used to analyze the expression patterns of the six genes.The results showed that Paom AG1 and Paom AG2 genes were expressed in both stamen and pistil;the two B genes,Paom PI1 and Paom PI2,were only expressed in perianths and stamen;Paom AP3 was found in bract,perianth and stamen.This result was consistent with the extended expression of the B gene in the original base angiosperm at the corresponding floral organ boundary,supporting the "boundary sliding" model.These genes showed a significant up-regulation tendency in the bud rapid elongation stage.The expression levels of the three B genes were significantly higher than those in the bisexual flowers.We speculated that the highly expressed B gene may be an important gene that leads to the development of male organs of P.omeiensis,and 2 C-Class gene may be involved in the development of pistil and stamen.4)The yeast two-hybrid technique was used to explore the interaction between proteins.The mode of action of most proteins were similar to that of their homologous proteins,but there were also some protein interaction models that differ from other results.Paom AP1 has a strong interaction with itself,Paom AG1,Paom AG2,and Paom AP3;3 B proteins do not form homodimer and only form a heterodime,in addition there is an interaction between Paom AP3 and Paom AG1,it showed that the evolutionary status of P.omeiensis is more primitive;there is no interaction between the C gene or itself.Combined with gene expression analysis,we infered the formation of tepals may be related to the Paom AP3 gene being heterologously expressed in the first floral organ and formed a heterodimer with Paom AP1.5)Through heterologous expression of B and C genes in wild-type Arabidopsis thaliana,it was found that the transgenic Paom AG1 wild-type Arabidopsis was significantly shorter and dwarf than the control,and rosette and cauline leaves were smaller and curled.It showed obvious early flowering,about one week earlier than that of the control,and the number of flowers on the inflorescence decreased,the "stigma" papillae appear at the tip of the sepal,and the petal development is abnormal,extremely reduced or even missing.This indicates that the Paom AG1 gene retains the function of the original class C gene and is involved in regulating the occurrence of carpels and inhibiting the activity of class A genes.Three wild-type Arabidopsis transgenic B-genes failed to show a homologous shift from the carpel to the stamen,but there were different degrees of phenotypic changes at the sepal.The wild type Arabidopsis which had been transfected with Paom AP3 gene showed a white-edged petaloid structure.The distance between the wild type Arabidopsis petals and the Paom PI1 and Paom PI2 genes was larger.We indicated that the three B genes have effects on the formation of petals,but their effects on stamen development need further study.