Comparison Analysis Of Some Differential Genes Between Precocious And Normal Trifoliate Orange [Poncirus Trifoliata (L.) Raf.] Based On The Whole Genome Resequencing

Citrus is one of the most cultivated fruit trees in the world, a long time of juvenile stage is needed during its growth and development, which greatly hindered the early access of citrus high yield and study of genetic breeding. Precocious trifoliate orange is early flowering mutant of wild trifoliate orange, they had nearly the same morphology characteristics except significant flowering habit, what’s more, previous studies found that they have the same genetic background, which make them become perfect materials for the studying on flowering-regulation mechanism of citrus. Our laboratory re-sequenced the whole genome of precocious trifoliate orange and trifoliate orange, and bioinformatics methods were used to analyze the re-sequenced data. We obtained a lot of sequence information that different between precocious trifoliate orange and trifoliate orange. My study focused on the InDel that exsited between the two materials and identified its correctness by experiments. In addition, we verify the function of 3 differential genes screened from identified work. The main results are as follows:1. We classfied the InDels existed in the CDS region. A total of 97 pairs of primers were designed using the Citrus clementina whole genomic data as a reference, then we verified the InDels by way of PAGE and AT clone sequencing;2.83 primer pairs showed clear bands in PAGE gel. Only 12 primer pairs showed diffence between precocious trifoliate orange and trifoliate orange, reflecting low accuracy by genome re-sequencing and analysis.40 primer pairs showed the same bands between the two speciese but differrent with clementina mandarin, illustrating the genetic differences between the trifoliate orange and orange which may provide reference for the development of molecular markers between trifoliate orange and orange;3.12 genes were identified there are InDels between precocious trifoliate orange and normal trifoliate orange, they are:Pt-21 (no comment).,Pt-35(highly ABA-induced PP2C), Pt-49(RING-H2 finger C2A),Pt-74 (Disease resistance protein), Pt-75 (Disease resistance protein), Pt-77(RLP1 Receptor like protein), Pt-81 (MES10 methyl esterase), Pt-83 (CCHC-type family protein), Pt-84 (NAD+), Pt-86 (MBD1), Pt-87 (no comment), Pt-88 (no comment);4.3 key differential genes which were named as PtAGO5, PtRZF and PtCCCHZF were selecteed for the next study. Gene expression analysis via RT-PCR showed that the expression of PtRZF and PtCCCHZF gradually increased during the self-cutting phase, indicating that the two gene may be involved in the citrus flowering transition; PtAGO5 showed the same expression patterns with the other two genes in spring shoots but not in summer shoots, may be related to the gene’s unique function;5. We constructed 6 over-expression vectors based on the 3 key differential genes, then transfored into the mordel plant of tabacco. Now we have obtained To generation of 35S::PtAGO5 transgenetic plants and transgenic tabacco callus of the other two genes, but no significient differential phynotype has been found.