Identification And Cloning Of Differentially Expressed Genes In Endosperm At Two Key Development Stages Of Different Maize Inbreds

Endosperm accounts for more than 80 percent of grain weight in maize. The development,proliferation and filling of endosperm cell play determined role in the formation of grain weight and quality.Although many studies about kernel endosperm have conducted,most of them were focused on the features of starch and the role of key enzymes in the process of starch synthesis in maize grain.Little is known about the development mechanism of grain endosperm in different periods, especially in the molecular level.In our present study two typical inbred lines for grain size in maize,a popcorn inbred N04 with very small grain and a normal dent inbred Dan232 with large grain,were chosen to reveal the differentially expressed genes in the key periods of endosperm development.After studying on the process of grain filling for the two inbreds,a large collection of ESTs were cloned,which were differentially expressed during two crucial periods of endosperm growth.Some ESTs were validated in expression,electronic mapped,analyzed in comparative genomics. A full-length cDNA segment was cloned,which was a GTP binding protein belonging to Arf family.Its expression situation was studied in the development of endosperm for the popcorn inbred N04 and the tissue characteristics were detected.These results were very helpful in revealing relative genes in the development of endosperm for different types of maize inbreds,and in understanding the molecular mechanism in endosperm development and grain filling.However,the most important objective was to find candidate genes for grain weight,to provide new gene resources,high level materials and theoretical basis for the improvement in grain weight and grain quality.The results were as follows:1.Richards function was used as a basic model to study the process of grain filling for the two inbreds,N04 and Dan232.The results showed that the N04 had higher initial growth potential,less greatest rate of grain filling and less time reaching the peak of grain filling than Dan232.Although the initial growth potential for Dan232 was smaller than N04,Dan232 had larger greatest rate of grain filling, average grain filling rate and 100-grain weight,and longer dynamic growth period than N04.This indicated that there were great differences in metabolic activity of the two inbreds during the process of grain filling.According to the fresh and dry weight of endosperm,embryo and pericarp in 10,15,20,25,30 and 35 DAP six stages,10 and 20 days after pollination(10 DAP and 20 DAP) were considered the key periods for the development of grain endosperm in maize.Microstructures of the two inbred lines in 3,5,7,10,15,20 and 25 DAP seven stages during endosperm development showed that endosperms were in the stage of cell division in 10 DAP,then endosperm cells accumulated substances rapidly.In 20 DAP,endosperm cells were filled with starch grains.The endosperm structure was also different between the two inbreds, hard and soft endosperm for N04 and Dan232,respectively.Therefore,10 DAP and 20 DAP were decided as two key stages for further study on the differentially expressed genes during endosperm development.2.Forward and reverse subtractive cDNA libraries were constructed using suppression subtractive hybridization(SSH) method,using 10 DAP and 20 DAP endosperms of the two inbreds.The four subtracted cDNA libraries enriched gene expressed specifically or at higher level in early stage and middle stage of endosperm development for N04 and Dan232,respectively.After verification through reverse Northern blotting,a total of 902 non-redundant cDNAs were obtained.The overlapping analysis for multiple sequences was conducted using BioEdit and ClustalW software.All 344 Unique ESTs contained 204 contigs and 140 singletons. According to Blastx search results,160 ESTs were obtained after excluding redundant sequences among the four subtracted cDNA libraries.It was inferred that their coding proteins had great homology with those amino acid sequences of known proteins in other species.Among those differentially expressed sequences,20%were unknown functions or could be considered as new ESTs.Those ESTs with known functions related to diverse functional categories,including basic metabolism,transcription and regulation,signal transduction,cell growth and division,protein processing and transporter,accounting for 20%,5.21%,2.6%,3%,26.04%and 5.73%,respectively.3.A total of 70 ESTs were located on the maize whole 10 chromosomes by in silico mapping and comparative mapping,of which 24.29%were located on chromosome 4,only 2.86%were located on chromosome 10.Nine ESTs(12.9%) were located on the same marker internals as those previously detected QTL for grain weight,which were related to five chromosomes,chromosome 1,2,3,6,7 and 8. Comparing with the QTL detected for 100 grain weight using the same inbreds,No4 and Dan232 as in this study,the chromosome locations of two ESTs,PE12C5 and PE15C3,were all the same as one QTL for 100 grain weight.These two ESTs might encode zinc-finger proteins and GTP-binding protein by inference.4.A full-length cDNA sequence of a putative GTP-binding protein was cloned using in silico cloning approach and was confirmed by RT-PCR.The sequence was 938 bp.The coding region was 609 bp,which encoding a protein with 202 amino acids.The 5' non-coding region was 237 bp,and the 3' non-coding region was 89 bp. Molecular weight of the deduced protein was 22.77 KD,with the isoelectric piont 6.13.The fore 15 amino acids were the signal peptide.The protein had a Arf family domain of a small G-protein gene,and a domain for the ABC transporter protein.In inbred N04,this gene showed high expression in early stage of endosperm development,and then gradually decreased.Also,this gene had different regulating role among various tissues,it expressed highest in the stem and the lowest in the root, but simultaneity expressed in root,stem,leave,and embryo with no tissue-specific expression.