Research Of Pericarp Developmental Rules And Functional Analysis Of AG-like Transcription Factors In Litsea Cubeba(Lour.) Pers.

Litsea cubeba（Lour.）Pers.（Lauraceae）,is a famous essential oil tree in China.L.cubeba flowers in January to March,the fruit becomes to enlarge and mature with diameter 5-6 mm.The colors of fruits change from green to red,and finally black in August or September,and then dropped off.L.cubeba contains essential oils in all tissues,especially in the pericarp tissues（3%-4%）.The production of essential oil from peels,is widely used in cosmetics,medicine,food and other chemical industry.It is valuable to investigate the mechanism into the development of L.cubeba fruits and peels.The knowledge on the pericarp development of L.cubeba would provide an important basis of the essential oil production.Here,we conducted the analysis of the endogenous hormone content,gene expression patterns and the functional verification on genes related to fruit development of L.cubeba.The main results include the following aspects.（1）ABA was significantly and highly expressed in the middle stages of L.cubeba pericarp development.We detected the endogenous hormones content in the L.cubeba tissues,The results showed that the contents of IAA were exhibited the highest level in the fruit tissues,then is the leaf,and low in flower tissues.The content of TZR was shown higher in the leaf compared with flower and fruit tissues.And the content of GA₁and GA₄were higher in the fruit tissues.The content of endogenous hormones in pericarp developing stages showed that JA was especially highly in the unfertilized ovary tissues,ABA was especially highly in the middle stages of pericarp development,and the content of GA₁was rising in the later stages of pericarp development.The result suggested that ABA may play an import role in L.cubeba’s pericarp development stages,and JA may play an import role in the initial ovary development,and GA₁may participate in fruit ripening phases.（2）The expression pattern of key genes during pericarp developing of L.cubeba.We then picked 12 phases of Litsea cubeba’s fruit and pericarp,from the unfertilized ovary to the matured fruits,and acquired 32 888 DEGs from the RNA-seq data.The expression clusters analysis of all DEGs showed that Cluster 4,which contained 457 DEGs,was enriched on MADS-MIKC transcription factors indicating that MADS-MIKC played an important role in peel development.The further investigation into the correlation between the gene expression and ABA contents pattern showed the positive correlated genes were 1 786,among them 136were TFs et al.,and the negative correlated genes were 583,and 102 TF et al.among them.（3）Lc Sca＿437.11 had an important role in the reproductive growth.A significant negative correlation was found between the gene of Lc Sca＿437.11 expression pattern and the contents of ABA.The expression of TALE family gene Lc Sca＿437.11 was increased with the pericarp development stages indicating the gene plays an important role in pericarp development in L.cubeba.The overexpression of Lc Sca＿437.11 in Arabidopsis induced later flowering in T₃generation,but no significant change was found in the size of fruits and the depth of pericarp.（4）Two AG-like genes were specifically expressed in pericarp of L.cubeba.The investigation into MADS-MIKC transcription factors indicated that two AG-like genes（Lc MADS31 and Lc MADS28）were highly expressed in L.cubeba pericarp developing stages.The q RT-PCR verification further indicated that Lc MADS31 has higher expression during the later period of L.cubeba fruits development,and Lc MADS28 was higher expressed in the middle period of L.cubeba fruits formation.The results suggested that Lc MADS31 and Lc MADS28 play an important role in fruit and pericarp development.（5）Two AG-like genes（Lc MADS31,Lc MADS28）in L.cubeba were involved in reproductive growth and fruit development.We further isolated the two AG-like genes（Lc MADS31,Lc MADS28）and conducted the sub-cellular location and functional verification.Both of these Lc MADS31 and Lc MADS28 were located in the nucleus.Over expression of both Lc MADS31 and Lc MADS28 could induce the advance of reproductive growth,early flowering and exert an obvious impact on the morphology of siliques in Arabidopsis,such as curly shorter siliques compared to wide type.The morphology was stable in T₃generation.The results illustrated that Lc MADS31 and Lc MADS28 were two functional genes,and can influence the reproductive growth in plants,like flowering time,fruit size and pericarp development.Moreover,the phenotype in over expressed Lc MADS31 and Lc MADS28Arabidopsis caused not only by the exogenous gene,but also synergistic effect of the endogenous genes,such as the expression of FUL,AG,STK.