Studies On The Molecular Mechanism Of MdCPRF2 Regulating The Gravity Fixed-point Angle GSA Of Apple From Indeterminate Roots Of Rootstock

Apple industry is a large industry in the development of agriculture,step by step towards the development of hundreds of billions of national advantageous characteristic industrial clusters on a new journey,the root rootstock is the best choice for the high standard garden at present.The Gravitropic set-point angle(GSA)from the anvil root is large,no gravitropic root,shallow distribution,the adaptability and resistance are poorer,they perform inferior in the drought barren orchard in our country,which restrict the quality and improve effect of the apple industry.Selecting and breeding deep-rooted apple rootstock with smaller GSA is beneficial to improve resistance,improve and stabilize tree potential,increase ground fixation of rootstock,and increase yield and economic benefit.According to their adventitious root GSA,the two varieties of apple autoroot stock selected in the early stage of this study were divided into shallow root type and deep root type.Studies have shown that auxin response factor Md ARF19(AUXIN RESPONSE FACTOR19),as a key factor regulating the formation of GSA in apple advanvil root,directly activates Md FLP(FOUR LIPS)and then participates in the regulation process of Md FLP determining the gravity-oriented fixed point Angle of advanvil root.In this study,a b ZIP(basic region/leucine zipper motif)family gene CPRF2(Common Plant Regulatory Factor2)was obtained through screening,which may regulate the expression of Md ARF19.The biological function and molecular mechanism of Md CPRF2 in the formation of GSA have been deeply explored.The main results are as follows:1.Apple Md CPRF2 gene and Md ARF19 gene were cloned from the c DNA templates of deep-rooted apple rootstock 13-22,9-26 and shallow apple rootstock 13-13,13-10,both of which were localized in the nucleus.Md CPRF2 has conserved b ZIP domain and is a member of the b ZIP gene family.It is closely related to Md CPRF2-like in Malus domestica,Atb ZIP63 in Arabidopsis and Pb CPRF2 in Pyrus bretschneideri.Md ARF19,as an auxin response factor,belongs to the ARF gene family and is closely related to Md ARF19-like in Malus domestica,Ms ARF19-like in Malus spectabilis,Pb ARF19-like in Pyrus bretschneideri,Md ARF7-like in Malus domestica and Pm ARF7 in Prunus mume.2.Using the RNA samples of root,stem,leaf,flower and fruit at different periods of 13-22,9-26,13-13 and 13-10 as materials,the relative expression level of Md CPRF2 gene was analyzed by q PCR biotechnology,and it was found that the relative expression level of Md CPRF2 gene was the highest in roots.and the expression level in deep roots was higher than that in shallow roots.It is suggested that Md CPRF2 plays a key role in the formation of adventitious root GSA of apple.3.Exogenous auxin treatment was applied to the advection roots of 13-22,9-26,13-13 and 13-10,and it was found that the expression of Md CPRF2 was induced by auxin.The induction of shallow root types 13-13 and 13-10 was the most significant at 2h,while that of deep root types 13-22 and 9-26 was the most significant at 4h.The results showed that the expression level of Md CPRF2 was significantly increased compared with the control after microgravity treatment of deep root type and shallow root type,and the content of IAA was significantly higher than that of the control under microgravity detection of different root types.The expression level of Md CPRF2 was up-regulated after dark treatment and restorative light treatment.The relative expression level reached the highest after dark treatment at 4h.The transcription level also recovered to 0h after restorative light treatment,and the expression fluctuation of Md ARF19 was similar to Md CPRF2.It was speculated that auxin treatment,microgravity treatment and transient dark treatment could induce the expression of Md CPRF2.4.The overexpressed transgenic strains of Md CPRF2-OX1,Md CPRF2-OX2,Md CPRF2-OX3 and Md CPRF2-OX1 were constructed respectively.Phenotypic analysis showed that when the roots of the transgenic strains started to appear,they showed faster downward growth and eventually showed smaller GSA compared to the no-load control.By q PCR,the expression level of Md ARF19 gene was significantly increased,and the endogenous IAA content was significantly increased compared with the control.The overexpressed transgenic strains were treated with microgravity,and the transgenic roots of Md CPRF2-OX5-M(shallow root type 13-13)and Md CPRF2-OX6-M(deep root type 13-22)were obtained,which did not maintain normal gravity-oriented growth.After microgravity treatment,the expression levels of Md CPRF2 and Md ARF19 genes were significantly increased,and the endogenous IAA content was significantly increased,but there was no significant difference in IAA content between shallow and deep root transgenic roots.At the same time,Md ARF19-OX5 and Md ARF19-OX6 overexpressed transgenic lines were constructed respectively,and the expression level of Md CPRF2 gene was not significantly different from that of the control.Therefore,the present study suggests that Md CPRF2 can positively regulate the expression of Md ARF19 and participate in the auxin regulation process,and its regulation of apple autoanvil advental root GSA requires the participation of gravity signal.5.In order to further study the function of Md CPRF2,the overexpressed Md CPRF2 vector was transformed into Arabidopsis Thaliana to produce transgenic Md CPRF2-OE1,Md CPRF2-OE2,Md CPRF2-OE3 and Md CPRF2-OE4.The number of initial and lateral roots increased in the transgenic strains.These results indicate that Md CPRF2 may be involved in root initiation.