SSH Library Construction Of The Semet From CMS-C Line And Its Maintenance Line In Maize

The use of heterosis was an effective way to improve crop yield per unit.Maize(Zea mays L.) was one of the world's first crops using heterotic.Use of male sterile lines of a hybrid system can be removed from artificial emasculation,lower labor costs and improved seed purity.In order to give better play to advantages of heterotic in the use of male sterile lines and approach the male sterile molecular mechanism in maize,the sterile line C48-2 and the maintain line N48-2 were used.We collected the tassel of the two materials that semet was occupying in the development of tetrad stage, monokaryotic stage and dikaryotic stage and then peelled out the semet.The Trizol method was used to extract RNA from the two materials in three development stage, then part balanced mix the total RNA of the sterile line and maintenance line in three different development stage,then used the magnetic bead separation and purification to mRNA.These two groups were reverse transcription to cDNA.We used SSH and T/A cloning techniques to build C48-2 and N48-2 forward and backward libraris.Dot blot technique was used to screen positive clone,through the sequencing to search differential expression genes in sterile line and maintenance line.Main results are as follows:(1) Through twice subtraction and twice PCR amplification,we obtained forward subtraction production that used the sterile line C48-2 as the tester when maintenance line N48-2 as the driver and backward subtraction production that used the maintenance line N48-2 as the tester when the sterile line C48-2 as the driver.Ligated the two group of difference fragment to the vector pMD 19-T,heat shock transform and combined the blu-white-spotting screen to construction forward SSH library containing 425 clones and backward SSH library containing 220 clones.(2) Random selected bacterium liquid from library to PCR amplification and detected out that the length of insertion elements were from 200 to 700bp,average length was about 300bp.(3) The PCR productions of bacterium liquid in the same library were denatured and dot on nylon membrane to make high density lattice membrane.Random priming was used to mark the cDNA ofC48-2 and N48-2 whit DIG.as a result,11 clones were acquired from the forward library,and 12 clones from the backward library.All selected clones were sequence by the sequence company.All sequences were deleted the primer and vector sequence by the software of DNAMAN,homology Blast to obtain 21 uni-ESTs sequence,in which include 10 from the forward library and 11 from the reverse library.(4) All of the sequences of insertion fragments were analyzed using bioimformatical methods.14 ESTs sequence will known about its functions,9 ESTs were unknown about its functions,including 1 new found ESTs.the differential expression genes related to functions of signal transduction,transcription and regulation,primary metabolism and second metabolism et al.The causes of the CMS were presumed by correlated informations of the functional gene in the research:(1) Ca²⁺ signal transduction anormalyOne Ca²⁺ signal transduction related gene was obtained in the research,the function of the gene was constraint Ca²⁺,it was down regulation expressing in C48-2, presume the short of the protein induced anomal release of Ca²⁺ and then locked the phosphorylation of CDPK,thus lid the pollen in no carbon source supply condition in development stage.This result was coincidence in the research of CMS-S in maize.(2) ApoptosisIn the plants,apoptosis participated many normal development and physiological process,in the development process of staminate flower,apoptosis of semet organism would cause male sterility.Earlier researches had indicated that gamete abortion of CMS-S was concerned with PCD in maize.In the research we obtained 4 apoptosis related genes.Aspartic proteinase nepenthesinâ…¡was decreasing expressed in C48-2, presume to BCL-2 gene.ogrammed cell death 2-lik,omega-3 fatty acid desaturase and ethylene receptor were up-regulation expressed.This three genes were important genes in apoptosis.(3) Starch related substance metabolism anomalIn staminate flower development process,the quantity and compositions of starch,sucrose and other contents in pollen were close correlated with pollen sterility.In the research,two genes related starch buildup.Alanine aminotransferase was the main enzyme to control the form of pyroracemic acid in glyconeogenesis and alanine inverted to pyroracemic acid was the key initiation in glyconeogenesis. Glucose-6-phosphate dehydrogenase was the key enzyme in pentose phosphate pathway.We presumed that the anomal expressing of these two enzyme was close correlated with male sterility.