Cloning And Expression Analysis Of Heterosis Related Gene SiDUF1618 In Millet

As one of the main miscellaneous grain crops in China,Millet(Setaria italica)has the characteristics of high nutritional value,strong drought resistance,stable and high yield,while hybrid millet has more prominent characteristics.Heterosis utilization,as one of the main ways to improve crop yield and resistance,has been widely used in breeding practice,but its genetic mechanism is still unclear,which restricts the further improvement of hybrid yield and research level.With the progress of contemporary biotechnology and the gradual deepening of heterosis research,researchers pay more and more attention to the mining of heterosis-related genes.In the early stage of this experiment,the booting spikelets of conventional millet varieties,strong dominant hybrids,medium dominant hybrids and weak dominant hybrids were collected in2018.The hybrids were compared with conventional varieties by RNA-seq to explore the differentially expressed genes related to heterosis.A certain number of differentially expressed genes related to heterosis were screened,and 15 heterosis related genes were cloned through a series of studies.On the basis of the above,I cloned a gene with DUF1618 domain,and explored and analyzed the role of SiDUF1618 in the formation of millet heterosis by bioinformatics analysis,subcellular localization and expression pattern analysis.The following are the results of this experimental study:(1)The CDS sequence of SiDUF1618 was cloned and three kinds of sequences were obtained: 01-seq,02-seq and 03-seq.01-seq is 1347 bp,encoding 448 amino acids,02-seq is 1512 bp,encoding 503 amino acids,03-seq is 1326 bp,encoding 441 amino acids.Sequence 01 and 03 have the highest similarity,and the proteins encoded by the three sequences belong to unstable proteins,all of which contain two low complexity region regions and one DUF1618 domain.(2)The subcellular localization of tobacco showed that 01 and 03 sequences were located in cell membrane and nucleus,while 02 sequence was located in cell membrane,which revealed that the three sequences might be involved in millet stress tolerance during the formation of millet heterosis.01 and 03 sequences may be involved in the regulation of millet gene expression.(3)Through the analysis of DUF1618 gene family in millet,117 members of DUF1618 family were identified.MEGA 7 software is used to analyze the evolution of 117 members of DUF1618 family which are divided into four groups.In group I,sequences 01,02 and 03 were located in the same evolutionary branch as KQK93854 and KQK93851,indicating that five genes may have evolved from the same ancestor.The chromosomal mapping of 117 members of DUF1618 family showed that all the members were distributed on 9 chromosomes of millet,and most of the genes were arranged in gene clusters.(4)Through GO enrichment analysis,15 DUF1618 family members may regulate the growth and development of millet through biological regulation,stress response,cell membrane components and enzyme catalytic activity.The genomes of wheat,rice and sorghum were analyzed by intergenomic collinearity with millet,respectively;12 groups of DUF1618 members which may be lineal homologous genes were obtained,indicating that with the evolution of Monocotyledon crops,some members of the DUF1618 family may replicate in the way of gene repetition among Gramineae genomes.(5)By analyzing the expression patterns of SiDUF1618 in leaves,ears and roots of different hybrids at booting stage and heading stage,it was found that SiDUF1618 was mainly expressed in the ear of weak dominant hybrid 3739 at booting stage.At booting stage and heading stage,the expression of SiDUF1618 in leaves,ears and roots of weak dominant hybrids 3739 was generally higher than that of other strong dominant hybrids and medium dominant hybrids,which is suggested that this gene may play a negative regulatory role in the formation of heterosis in millet.(6)Through the analysis of the promoter of SiDUF1618,it contains ABA,MYB,Me JA and auxin response elements.The leaves of millet at seedling stage were treated with ABA(50 μM),Na Cl(50 m M),Me JA(50 μM),IAA(10 μM)and 6-BA(10 μM);then the leaves treated for 0 h,1 h,2 h,3 h,4 h and 6 h were sampled.The expression of SiDUF1618 under hormone treatment was analyzed by real-time quantitative PCR,and it was found that the expression of SiDUF1618 decreased under ABA and Na Cl treatment,while it was up-regulated by Me JA treatment,indicating that SiDUF1618 may play a role in millet defense response,while IAA and 6-BA treatment increased its expression,indicating that it may be involved in millet auxin signal pathways..