| Litchi has been cultivated for a long history and kept a lots of ancient litchi trees in Fuzhou,which are of the important historical,cultural and scientific research values. In order to betterprotect Fuzhou ancient litchi trees and mine the resistance gene resources. This research optimizedthe conditions of the in vitro minimal growth conservation of EC in Fuzhou ancient litchi trees,and further saved the main ancient litchi tree resources of Fuzhou, which focused on the pastresearch foundation of the laboratory; cloned the three types of SOD genes from ancient litchitrees EC, and analysed the expression of SOD genes in the process of in vitro preservation, and thechanges of SOD activity of ancient litchi trees EC under the low temperature stress. The mainresults were as follows:1The optimization of conditions of in vitro minimal growthconservation of EC in Fuzhou ancient litchi trees1.1The induction and preservation of the embryogenic callus in ancient litchitreesThis study first induced the EC in ancient litchi trees ã€X1, X2("songli"), X3, X6, X8, X9,X11, X12, X13, X14, X15, X16, X17, X18, X19and X20ancient litchi trees in Xichansi Temple,W3and W4ancient litchi trees in Wushan Hill, H3ancient litchi tree around Huaqiao Bulding】,the results showed that the induction of the EC closely related to the types, concentrations andcombinations of Plant hormones, except the induction rates on M1of X11and X19were higherthan on M2, the rest ancient litchi trees' anther induction rates on M2were higher than those onM1. On the same culture medium, the induction rates of EC in different ancient litchi trees weredifference, the induction rate of X11on M1was79.55%, and the induction rate of H3on M1was0.00%. The EC which just induced had strong ability to produce embryos, the number of embryosin the same ancient litchi tree were difference in different culture media.After the EC which just induced trained alternately on M1and M2for four generations in thedark, they could be conserved alternately for a long time on the media (M3, M4), which hormoneswere half of M1and M2, under the low light. X12, X14, X15, X16, X17also had more ofembryos after conservation for many generations.1.2The optimization of conditions of in vitro minimal growth conservation of ECin ancient litchi trees The EC of X2was as the test materials, orthogonal experiment was designed based on sucrose,mannitol, inositol, and temperature, to study the in vitro restricting conservation in ancient litchitrees. The results showed that the best storage condition for X2EC was on the MS medium with2,4-D1.0mg.L-1, sucrose20g.L-1, mannitol20g.L-1, inositol0.3g.L-1, agar7g.L-1, with the pHvalue of5.8, at19℃in darkness.Low temperature culture conducive to improve the cell viabilityof embryogenic callus in X2; the growth rate of X2EC was negatively correlated with sucroseconcentration, but improving sucrose concentration could increase the growth of X2EC.2In vitro conservation of the main germplasm resources of ancientlitchi trees in FuzhouThe conservation results of different ancient litchi trees EC in the best conservationconditions(G7) showed that there were more embryos produced in the conservation process of X6,X8, X12, X14, X15, X16, X17, just could conserve about100d, the others grow well, couldconserve for120d, and40,44and H3could conserve longer,135d; the growth of33,40and44in the conservation process were more than most of the new EC, all the22different ancient litchitrees EC could conserve normally in the best conservation conditions(G7).3Cloning of the three types of SOD genes from EC in Fuzhou ancientlitchi trees11members of complete cds and a partial cds of the three types of SOD genes weresuccessfully cloned from EC in Fuzhou ancient litchi trees by RT-PCR combined with RACE.3.1Cloning of Fe-SOD genes from EC in ancient litchi treesEight members of Fe-SOD complete cds and a partial cds were cloned from EC in ancientlitchi trees, among them, LcFe-SOD2(JQ771621) was the partial one with885bp; and the mRNAsfull-length of LcFe-SOD(JN671967.2), LcFe-SOD1(JQ771618) and LcFe-SOD5(JQ861697) wererespective1176bp,1205bp and756bp, and respectively coded236,174and159amino acids;LcFe-SOD1A(JQ861693),LcFe-SOD1B(JQ861694),LcFe-SOD3(JQ861695),LcFe-SOD4(JQ861696) and LcFe-SOD6(JQ861698) were all ORF, the ORFs of LcFe-SOD1A and LcFe-SOD1B wereall525bp, coded the same protein with LcFe-SOD1; the ORFs of LcFe-SOD3, LcFe-SOD4andLcFe-SOD6were respective525bp,414bp and711bp, and respectively coded174,137and236amino acids; LcFe-SOD1and LcFe-SOD4were splicing mRNA variants with the same splicingposition in gene ORF, and made the original protein translation change. But their splicing modewere different, respectively belonged to intron kept splicing mode and exons were splicedselectively mode.According to bioinformatics analysis, all of the proteins encoded by Fe-SOD genes were acid,unstable and closed to water, with a winded helix and without the signal peptide and transmembrane domain. The proteins encoded by LcFe-SOD and LcFe-SOD6may located inmicrobody (peroxisome), and the proteins encoded by LcFe-SOD1, LcFe-SOD3, LcFe-SOD4andLcFe-SOD5all may located in cytoplasm; the phosphorylation of all the proteins encoded byFe-SOD genes, which belonged to the family of Mn/FeSOD, occurred on serine, followed bytyrosine residues, among them, the proteins encoded by LcFe-SOD and LcFe-SOD6had the sameconserved domains of N-end and C-end, and four domains that exclusively belonged to the familyof MnSODISMTASE; the proteins encoded by LcFe-SOD1and LcFe-SOD3had the sameconserved domains of C-end and two domains that exclusively belonged to the family ofMnSODISMTASE, without the conserved domain of N-end; the protein encoded by LcFe-SOD4only had conserved domains of C-end, without domain that exclusively belonged to the family ofMnSODISMTASE; the proteins encoded by LcFe-SOD5only had the conserved domains ofC-end and two domains that exclusively belonged to the family of MnSODISMTASE.3.2Cloning of Mn-SOD genes from EC in ancient litchi treesThe ORF of Mn-SOD gene was cloned from EC in ancient litchi trees with665bp and coded221amino acids, named as LcMn-SOD, which had initiator codon (ATG) and terminator codon(TGA). According to bioinformatics analysis, protein encoded by LcMn-SOD was alkaline, stableand closed to water, without the signal peptide, winded helix and transmembrane domain, andmay located in the Mitochondria matrix space, peroxisome, mitochondrial inner membrane andmitochondrial intermembrane space, the phosphorylation levels of serine, threonine and tyrosinewere similar, and belonged to the family of Mn/FeSOD with the conserved domains of N-end andC-end, at the same time, the protein encoded by LcMn-SOD had five domains that exclusivelybelonged to the family of MnSODISMTASE.3.3Cloning of Cu/Zn-SOD genes from EC in ancient litchi treesTwo members of Cu/Zn-SOD gene complete cds were cloned from EC in ancient litchi trees,and were respectively named as LcCu/Zn-SOD1(JQ771619) and LcCu/Zn-SOD2(JQ771620), themRNAs full-length of them were677bp and700bp, with initiator codon (ATG) and terminatorcodon (TAA) and without3'UTR. LcCu/Zn-SOD2was splicing mRNA variants with the splicingposition in5'UTR, and didn't make the original protein translation change, belonged to intronkeep splicing mode. Both of them encoded the same protein which was acid, stable and closed towater, without the signal peptide and winded helix and have two transmembrane domain, locatedin the chloroplast and mitochondria, the phosphorylation levels of serine and threonine were thesame, and phosphorylation didn't occurred on tyrosine residues, and had four domains thatexclusively belonged to the family of Cu/Zn-SODISMTASE, and the binding site ofcharacteristic1and characteristic2that belonged to Cu/Zn-SOD. 4The expression variation of SOD genes in the conservation processof embryogenic callus in ancient litchi treesGAPDH as reference gene in this experiment, analysed the expression variation of LcFe-SOD,LcMn-SOD and LcCu/Zn-SOD1under the optimal preservation conditions (G7) and the normalstorage conditions (M3) by qPCR, the results showed that at the stage of save pre (10-30d),LcFe-SOD, LcMn-SOD and LcCu/Zn-SOD1common regulated the growth of EC both in G7andM3; at the stage of save middle (30-50d), the growth of EC was regulated by LcFe-SOD andLcCu/Zn-SOD1both in G7and M3; at the stage of save later (50-90d), the growth of EC mainlyrefulated by the coordination between different types of SOD, to maintain the dynamicequilibrium of SOD in embryogenic callus in ancient litchi trees. LcFe-SOD and LcCu/Zn-SOD1played a key role in G7conservation process.5The changes of SOD activity of EC in ancient litchi trees under thebest conservation conditions and ordinary conservation conditionsOn the base of the best conservation conditions of G7, we further discussed the changes ofSOD activity of EC in ancient litchi trees under the best conservation conditions and ordinaryconservation conditions. The results showed that SOD activity of ancient litchi tree X2droppedsmoothly and then rose, after dropped quickly to the lowest the SOD activity rose smoothly to thetop, finally dropped smoothly under the best conservation conditions with the extend ofconservation time. In the ordinary conservation conditions, SOD activity of ancient litchi tree X2rose slowly to the top before the30d, then dropped quickly with the extension of conservationtime.The peak of SOD activity was delayed under the best conservation conditions, and thestorage life of EC in ancient litchi trees was also lengthened.6The changes of SOD activity of EC in ancient litchi trees under thelow temperature stressThe results of the changes of SOD activity of EC in ancient litchi trees under the lowtemperature stress showed that, the SOD activity of EC of ancient litchi tree X2was the highest at20℃, and the temperature of5℃was the cold damage critical temperature of X2, lowtemperature obviously restrained the SOD activity of EC of ancient litchi tree X2; X11had strongability to resist cold, but it's ability to resist coldness was lower than the ability to resist coldnessof other ancient litchi trees;40, W3, W4, H3,44, X2and hy30might have the similar coldresistance types. |
