High-Troughput Screening And Analysis Of Genes Related To Anther Development In Oilseed Rape (Brassica Napus L.)

Anther development is a complex process composing many biological events and is a highly ordered developmental process.The different developmental stages of anther growth involved a large number of genes orderly expressed.Brassica napus,one of most important oil crop and had advange in exhibiting a great deal of hybrid vigor.However,our knowledge about the mechanism controlling early flower and anther development of Brassica napus is still limit.Microarray method is a powerful technique used to detect change of genes expression profiles on a genome-wide scale and to study the gene regulatory networks and molecular mechanism of development.So it is necessary for us to make use of the microarray to study the different developmental stages of Brassica anther.In the present study,we collected anther at three different developmental of Brassica napus namely,small flower buds of<1 mm in length(SB,at pollen mother cell stage),anther from medium buds 1-3mm in length anther(An-MB,from tetrads of micropores to late uninucleate micropores stages),anther from large buds>3 mm in length(An-LB,late uninucleate micropores stages to mature pollen stage)and leaves(Le)were collected.We studyed the transcriptome of different developmental stages of Brassica napus anther,Zhongshang 9 using Agilent Brassica Oligo Microarray(4x44K)Genechip and analysed the distribution of differentially expressed genes between different developmental stages of anther compared with leaves and different developmental stages of anther compared with each other.In addition,we also analysed the differentially expressed genes of different developmental stages of Brassica napus anther to identify co-regulated genes and functional classification and significant metabolic pathways.Finally,qRT-PCR was used to verify the results of the gene chip.The major results obtained are as follows:(1)It was indicated that 30509 transcripts were expressed in leaves,32443 in SB,33300 in An-MB,32383 in An-LB.These data showed that the number of transcripts were similar in different developmental stages of anthers,but slightly higher than that in leaves,and the number of transcripts in An-MB was the largest.(2)Our analysis showed that 28279 transcripts were consitiutively expressed in four tissues and 1520 transcripts present in the SB,An-MB and An-LB samples except in Le.In comparison,the number of specific transcripts in Le,SB,An-MB,An-LB were 402,466,468,963,repectively.These specific transcripts that expressed in anthers and only expressed in three related anther samples but not in leaves may be the genes that related to anther development.(3)To identify genes expressed differentially among different developmental stages of Brassica anther napus,a stringent significance threshold:P-value<0.001 and Fold change>2 were used to screen the differentially expressed genes.The results showed that a total of 11678 transcripts were differentially expressed in at least two tissues examed.It was found that there were 8704 differentially expressed transcripts when compared SB,An-MB,and An-LB with leaves.Among which,4098 transcripts were up regulated in at least one different developmental anthers and 66 transcripts were up regulated in all three developmental anthers.4643 transcripts were down-regulated in at least one different developmental anthers and 672 transcripts were down-regulated in all three developmental anthers.With anther development,there was an increase in the number of differentially expressed genes between anther and leaf tissue.In addition,when SB,An-MB and An-LB compared with each other,it was showed that the total number of differentially expressed transcripts were 5939 and there were 2496 up-regulated transcripts and 3465 down-regulated transcripts.(4)The DAVID online software were used to analyze the functional category of all the differentially expressed genes.GO analysis showed 87,61 and 114 signifieantly up-regulated GO in SB,An-MB,An-LB as compared with leaves.There were 16 up-regulated GO in An-MB as compared with SB.And 51 up-regulated GO were present in An-LB when compared with An-MB.There were 116 up-regulated GO in An-LB as compared with SB.The pathway of differentially expressed genes of different developmental stages of anther and leaves compared with each other were analyzed using KOBAS.In comparasion with Le,pathway analysis showed 2,3 and 7 significant pathways of up-regulated genes in SB,An-MB,An-LB(P-value<0.05 and FDR<0.05).The significant pathways of up-regulated genes in An-MB as compared with SB were 2.There were 7 significant pathways of up-regulated genes in An-LB as compared with SB.The significant pathways of up-regulated genes in An-LB as compared with An-MB were 3.(5)11678 differentially expressed transcripts were grouped into 30 clustering using K-means clustering algorithm according to their expression pattern in Le,SB,An-MB,An-LB.The gene number of each cluster ranged from 89 to 844.Cluster 1,3,5,6,11,12,27,29 contained a total of 1281 differentially expressed genes that significantly up-regulated in at least one developmental anthers,indicating that these genes is closely related to anther or pollen development,or play an important role in anther development process.Among the different expression pattern,some genes significantly up-regulated in SB,some in An-MB,some in An-LB and some significantly up-regulated in all three different developmental anthers.The genes related to anther development involved in pollen exine formation,pollen tube growth,transcription factors and fatty acid synthesis.(6)Based on the cluster analysis of differentially expressed,eight genes randomly selected from different cluster were chose to quantify their expression level by quantitative real-time fluorescence PCR(qRT-PCR)to verify the reliability of the microarray data.The result showed that the data of qRT-PCR were consistent with the data of microarray.This study screened a large number of genes related to development of oilseed rape anthers on a large scale.These results will provide important basic information for further isolating and identifying genes related to anther development and studying molecular mechanisms and regulatory networks of Brassica napus anther.