RNAi expression vector and construction of the self-incompatibility SFB gene from the‘Xiaobaixing' (Prunus armeniaca) apricot

Abstract: 【Objective】‘Xiaobaixing'(Prunus armeniaca) apricot is a characteristic and famous cultivarin the southern area of Xinjiang province. Its fruit matures early in mid-June with an oval shape and trans-parent light yellow color,hairless and smooth surface. While the‘Xiaobaixing'apricot displays the typi-cal S-RNase-based gametophytic self-incompatibility(GIS) controlled by S-allele,it also contains a pollen determinant and a style determinant,which help to mutually determine the self-compatibility or self-incompatibility of the apricot. The self-incompatibility of the apricots increases the labor costs during theproduction process and also increased the complexities of the cultivation process. The self-compatible(SC) apricot cultivars are important in promoting the industrial development of these characteristic apri-cots in Xinjiang. The SFB(S haplotype-specific F-box protein) gene is a good candidate for pollen-S determinates in Prunus species. Some SC apricot cultivars have been introduced and identified,the loss ofthe pollen-S function in Prunus SC mutants may be associated with deletion,insertion,and substitutionat the upstream area of the hypervariable regions in SFB genes when being compared with the SI(self-in-compatible) varieties. These result in a truncated protein without the expected benefit of the hypervariableregions,the abnormity of the hypervariable regions leads to an afunctional SFB and SC mutation. Manipu-lation of the hypervariable regions or its upstream area for the SFB gene,through using RNAi technolo-gy,will be a theoretically feasible method to regulate self-incompatibility in apricots at the molecular lev-el and allow for the breeding the SC apricot cultivars.【Methods】The RNAi plant expression vector of the SFB gene is constructed by applying the fusion PCR approach combined with the enzyme digestion con-nection method. Based on the results of the structural analysis for the 3'end full length c DNA of the SFB gene obtained from the self-incompatible apricot‘Xiaobaixing',we selected a 29 bp fragment as the in-terference target,which is 61 bp from the initiation codon located upstream from the variation area in the SFB gene,and also we selected a 242 bp fragment from the upland cotton(Gossypium hirsutum) genomicDNA sequence as the interval fragment to ensure that there would be a silence efficiency for the sequen-tial studies. The key to constructing the ihp RNA structure using the fusion PCR is the design of the PCRoverlapping primers;the adapter primers of the three fragments should include the“initiation”and“over-lapping”sections. According to the principle of the RNAi expression vector and construction of the inter-ference target,3 pairs of primers,which are overlapping primers,are designed by using Primer Premier5.0 software for developing the first round of PCR reaction and to clone the opposite,interval and positivefragments,which constitute the RNAi component of the SFB. Then the opposite,interval and positivefragments,after purification and concentration,are fused by applying the second round PCR reactionwithout primers for the ihp RNA(intron-containing hairpin RNA) construction. After recovery and purifi-cation,the largest recombinant fragments(ihp RNA contained) of the second round PCR are recombinedwith the cloning vector p EASY-T1 and transformed into E. coli DH5α competent cells. The correct recom-binant plasmids,which contain ihp RNA,are named p EASY-RNAi-SFB,after sequencing and analysis.The ihp RNA contained fragments are digested by the corresponding restricted enzymes(Xba I and Kpn I)and inserted between the Ca MV 35 S promoter and the NOS terminator of the plant expression vectorp CAMBIA- 35S-MCS-NOS- NPTII. The correct recombinant plasmids,after enzyme digestion (Xba Iand Kpn I) and PCR identification,are transferred into Agrobacterium tumefaciens LBA4404 by usingfreeze/thaw application. The positive colonies are screened and cultured on a solid YEB (Yeast Extract Broth) medium containing 125 g·L^-1 Sm (Straptomycin) and 50 g·L^-1 Km (Kanamycin) at 28 ℃. A singlecolony is chosen and then shaken(180 r·min^-1) at 28 ℃ in the YEB liquid medium containing the sameantibiotics with using the same concentration,then they are verified by using the corresponding specificprimers PCR identification.【Results】Sequencing results demonstrate that the 3'-end full-length c DNAof the SFB gene which we have obtained,has the basic structure of the SFB gene,consisting of a 912 bpcomplete ORF,an F-box domain composed of 42 amino acid residues at the N-terminal,two variable re-gions V1,V2 and two hypervariable regions HVa,HVb are included,with the V1 being located at thedownstream of the F-box domain,while the others are located at the 3'-end. The ihp RNA of the SFB areconstructed by fusing the opposite,interval and positive fragments with the second PCR reaction. The op-posite,interval and positive fragments were cloned with the self-designed primers after the first roundPCR reaction,and allowing for the 300 bp target fusion PCR products(ihp RNA contained fragments) ofthe component fragments to be obtained. The sequencing results showed that opposite,interval and posi-tive fragments were fused and we were able to successfully construct ihp RNA with an 29 bp hairpin armand 242 bp loop of self-incompatibility SFB in the‘Xiaobaixing'apricot without a self-intron. The re-sults of the digestion and bacteria liquid PCR prove that the RNAi component of SFB has been recom-bined with p CAMBIA-35S-MCS-NOS-NPTII. The results of the bacteria liquid PCR and sequencingprove that the RNAi vector can be successfully introduced into the Agrobacterium tumefaciens competentcells LBA4404.【Conclusion】The results prove that fusion PCR combined with enzyme digestion is feasi-ble for constructing the RNAi plant expression vector of the SFB gene. This creates a condition for RNAito regulate self-incompatibility in fruit trees. The traditional enzyme digestion connection method is pri-marily used in the construction of the RNAi expression vector in other types of fruit trees;the efficacy ofthe gene-silencing of the RNAi expression vector with ihp RNA is higher in the different types of hairpinstructure. However,the traditional process for ihp RNA is complicated and creates a long cycle. FusionPCR is a method that allows for the construction of the structure of ihp RNA by using PCR with overlap-ping primers. The ihp RNA could be immediately generated by PCR,rather than through construction ofan intermediate vector. It's easy to operate and no special restrictions are required for the target frag-ments compared to the traditional method. You can also fuse any fragments that you find interesting,andthe ihp RNA constructed by fusion PCR can be ligated with any of the plant expression vectors that youneed. The introduction of an interval region,such as the cotton genomic DNA fragment which is the in-tron sequence of Gh HSD1(11-β-hydroxysteroid dehydrogenase) from upland cotton(Gossypium hirsutum),could greatly enhance the stability of the inverted-repeat sequences in bacteria,and facilitate thecloning process. Increasing the molar concentration of the target fragments for fusion and the molar ratioof ihp RNA and the expression vector could accelerate the construction progress of the RNAi expressionvector. The Kanamycin resistant gene NPTII(Neomycin phosphotransferaseⅡ) and reporter gene GUS(β-glucuronidase) in p CAMBIA-35S-MCS-NOS-NPTII can be directly used for screening and identifica-tion of the transgenic plants. The RNAi expression vector of SFB will provide a good basis for transform-ing the Prunus armeniaca L.‘Xiaobaixing'apricot and contribute to molecular breeding of SC apricot cul-tivars. The sense and antisense expression vector of the SFB gene are constructed in our laboratory at thesame time,and used for comparing the inhibition efficiency of the antisense and RNAi expression vectorof SFB in transgenic apricots for further study. The combination of overexpression(by using the sense ex-pression vector) and RNAi technology could further reveal the functions of the target genes and their func-tional networks.