| The gene expression regulations mediating by micro RNA play vital roles in plant growth and development.Argonaute(AGO)proteins,as important functional proteins in the pathway of micro RNA related gene silencing,which were widely involved in the plant growth regulation and stress response.At present,there are still very few reports about AGO in wheat,much less systematically analysis on AGO gene families.In this paper,we studied the expression characteristics of AGO in the process of wheat growth and development,and preliminarily explored the function of AGO protein using VIGS mediated gene silence technology.This research will lay a foundation for further exploration on the mechanism of AGO related gene expression and regulation in the growth and development of wheat.The main results are as follows.1.17 TaAGO family members were indentified from wheatusing bioinformatics methods,and the A,B and D homologous sequences for each member were analyzed.Chromosomal localization revealed that these 17 members were distributed on 7 chromosomes of wheat,and most of the AGO members distributed on 2,6,7 Chromosome groups.In particular,three homologous genes of TaAGO1b(TaAGO1b-4A/-7A/-7D)are not located on the same chromosome group.Genome structure analysis showed that introns number of TaAGO gene family was 20~23,while TaAGO2 a and TaAGO2 b contained only 2 introns.Three homologous sequences of A,B,and D in a TaAGO genes are also different in the number and size of the introns.For example,the intron number of TaAGO10a-7A,TaAGO10a-7B,TaAGO10a-7D is 22,21,22,respectively.The size of the first intron of TaAGO10a-7B and TaAGO10a-7D is 8161 bp and 7543 bp,respectively,compared with 92 bp of TaAGO10a-7A.Further analysis indicated that three TaAGO10 a homologous sequences were differences in gene structure,yet it encoded amino acid sequence was highly similar.As compared with TaAGO10a-7A,the amino acid similarity of TaAGO10a-7Band TaAGO10a-7D is 94.4% and 96.7%,respectively.The relationship between the the first intron and the itsexpression will be studied.The amino acids numbers of 17 TaAGO proteins were between 692 and 1192,and the corresponding molecular weight were 78.40 to 131.42 KDa,with isoelectric point 8.64 to 10.05.All the TaAGO proteins were hydrophilic.Subcellular localization showed that 17 TaAGO proteins were located in the cytoplasm and nucleus,while the other 2 AGO proteins(TaAGO10 a and TaAGO10b)were located in the cytoplasm and mitochondria.2.Phylogenetic and cluster analysis showed that 17 TaAGO family members could be divided into three subclasses.This is consistent with the Arabidopsis AGO gene family classification(Clade ??Clade ?and Clade ?).10 TaAGO members(TaAGO1 a,TaAGO1 b,TaAGO1 c,TaAGO1 d,TaAGO5 a,TaAGO5 b,TaAGO5 c,TaAGO10 a,TaAGO10 b,TaAGO18)were clustered together as A subclass.B subclass contained 9 menbers(TaAGO2a?TaAGO2b?TaAGO7),C subclass contained 4 menbers(TaAGO4a?TaAGO4c?TaAGO4b?TaAGO6),shows that there is no difference in the evolution of the AGO family between monocotyledonous and dicotyledonous plants.3.The tissue expression of 17 TaAGOs in 5 wheat tissues or organs showed that 27 homologous loci of TaAGOs(TaAGO1c-6A/-6D?TaAGO1b-2A/-2B/-4A/-7A/-7D?TaAGO1d-7A/-7B/-7D?TaAGO4c-1A/-1B/-1D?TaAGO2a-2A/-2B?TaAGO2b-2A/-2B/-2D?TaAGO4b-7D?TaAGO4a-3A/-3B/-3D?TaAGO10a-7B/-7A)had highly expression levels in all tested tissues(seeds,spikies,leaves,roots and stems);The 7 homologous loci of 4 TaAGOs(TaAGO1b-2D?TaAGO2a-2D?TaAGO5c-5A/-5B/-5D?TaAGO10b-6A/-6B)family members are expressed only in spikes;The two family members TaAGO5b(TaAGO5b-2A/-2B/-2D)and TaAGO18(TaAGO18-3A/-3B/-3D)had the highest expression levels in seeds and lower expression levels in spikes.The results indicated that TaAGOs family members plays different roles in different wheat tissues.4.Full c DNA sequences of TaAGO1 b and TaAGO4 a were amplified in wheat JING 841 and Chinese Spring,and their A,B,D three homologous were isolated and identified.TaAGO1b-4A/-7A/-7D has similar genes structure,containing 21 introns.The gene length of TaAGO1b-4A/-7A/-7D was 3069/3099/2876 bp(a 2607 bp ORF),encoding 868 amino acids,with the predicted molecular weight 97.93/97.89/ 97.87 KDa and the isoelectric point 9.31/9.28/9.31.Amino acid sequence alignment showed that the similarity of amino acid sequence of TaAGO1 b gene in two wheat varieties was 98.5%,and there were 11 amino acid differences.As for TaAGO1b-4A,the 13 th,479th,770 th amino acids were different in two varieties.The 3th,158 th,179th,441 th and 546 th amino acids of TaAGO1b-7A were different,and the 334 th,410th amino acid of TaAGO1b-7A were different.TaAGO4a-3A and TaAGO4a-3D has similar genes structure too,containing 20 introns.The length of TaAGO4a-3A was 3040 bp(a 2748 bp ORF),encoding 915 amino acids,with the predicted molecular weight 101.77 KDa and the isoelectric point 9.31;The length of TaAGO4a-3D was 3012 bp(a 2751 bp ORF),encoding 916 amino acids,with the predicted molecular weight 101.93 KDa and the isoelectric point 9.06.Amino acid sequence alignment showed that the similarity of amino acid sequence of TaAGO4 a gene in two wheat varieties was 99%,and there were 8 amino acid differences.As for TaAGO4a-3A,the 5th,13 th,178th,423 th amino acids were different in two varieties.Compared with TaAGO4a-3A,TaAGO4a-3D was short of a proline(P)in wheat JING 841.Based on conservative domain analysis,the TaAGO1 b gene contained the typical conservative domain PAZ(207th~321th amino acids)and PIWI(497th~818th amino acids)of the AGO family,The location of PAZ and PIWI conserved domain of TaAGO4 a gene were 324th~339th and 568th~875th amino acids,respectively.5.The expression patterns of TaAGO during the seed germination were analyzed,which had similar expression pattern in the embryo and endosperm between Jing 841 and Chinese Spring,while the patterns of expression in the embryo and the endosperm are different.In embryo,TaAGO1 b gene was highly expressed in at 6 h,24 h and 72 h.In endosperm,TaAGO1 b gene was highly expressed in at 12 h,24h.As for TaAGO1 b,the expression level of TaAGO1b-7D washighly,while that of TaAGO1b-4A and TaAGO1b-7A were low,which indicated that TaAGO1b-7D might play important role during the seed germination.As a whole,the expression of TaAGO1 b was up-regulated during seed germination,involved in the germination process of seeds.the different expression patterns of TaAGO4 a were found in two wheat varieties.The expression pattern of TaAGO4 a in embryo and endosperm was similar in JING 841,with persistent up-regulated expression at 6-24 h,and maintained a high expression amount in following hours.In the embryo of Chinese Spring,the expression lever of TaAGO4 a was higher in the early stage of seed germination,while it was higher expressed in the endosperm during the late seed germination stage.The expressed differences of TaAGO4 a in two cultivars might implied that it played a regulation function during the process of seed germination?The expression of three homologous genes of TaAGO4 a was also different.The expression of TaAGO4a-3D was the largest in Jing 841,while the maximum expression of TaAGO4a-3B was observed in Chinese Spring,indicating that homologous genes might play different roles during seed germination.The expression of TaAGO in vernalization process was analyzed.For Jing,the expression level of TaAGO1 b was up-regulated expression in the middle and late stage of vernalization and its expression reached the maximum value at 24 d,increased by 5.4 fold.For Chinese Spring,The expression of TaAGO1 b gene was higher in the middle and early stage of vernalization and the expression level was the highest at 25 d,increased by 21 fold compared with the control.The TaAGO4 a expression patterns of two varieties were different.In Jing 841,the expression of TaAGO4 a was up-regulated at 6d,showing the highest value during 21d-27 d.However,the expression of TaAGO4 a increased rapidly in the early stage of vernalization treatment,reaching the highest level at 21 d,and then its expression decreased rapidly.Overall,compared with winter wheat,the up-regulation expression of TaAGO1 b and TaAGO4 a genes during vernalization process in spring wheat was faster and lasted for a long time.But the up-regulation expression amount of TaAGO1 b and TaAGO4 a genes was less than those in winter wheat.These different responses to vernalization indicated that TaAGO1 b and TaAGO4 a played individual roles in vernalization.6.Barly Strip Mosaic Virus Vectors containing TaAGO1 b and TaAGO4 a genes were successfully constructed and amplified using in vitro transcription technology.These virus vectors were inoculated with two-leaf stage wheat by smearing on leaf.At the 7th day,photobleaching phenomenon was occurred in inoculated plants.Based on Quantitative Real-time PCR analysis,at 7 days after inoculation,the expression of endogenous TaPDS gene decreased by 26% compared with control,and expression of endogenous TaAGO1 b and TaAGO4 a gene decreased by 20% and 25%,respectively.At 21 days after inoculation,the expression of TaPDS decreased by 62%,with TaAGO1 b and TaAGO4 a decreasing by 41% and 40%,respectively.Although no significant phenotypic changes were observed in the inoculated wheat,the expression of endogenous genes TaAGO1 b and TaAGO4 a were successfully inhibited by inoculation.In order to investigate the physiological and biochemical mechanism of TaAGO1 b and TaAGO4 a in the process of wheat growth and development,the RNA interference vectors of TaAGO1 b and TaAGO4 a were constructed.Based on the Agrobacterium mediated transformation method,we have successfully obtained some seeds of T0 generation transgenic wheat.Furthermore,molecular identification and screening on T1 generation transgenic wheat were in progress. |
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