To extensively understand the sequence feature and evolution of heat shock transcription factors (Hsf)in the genome of apple,fifty MdHsf genes were identified using bioinformatics methods at the whole-genome level of apple,and a series of analysis including sequence characterization,phylogenetic relationship,gene expression and selective pressure of MdHsf genes were further performed.Phylogenetic relationship and sequence characterization analysis showed that,like the model species Arabidopsis and rice,50 MdHsf genes were divided into three subfamilies A,B and C.Additionally,at least two genes were found in the same end clades in the phylogenetic tree,indicating that the lineage-specific amplification had happened during evolutionary processes of apple Hsf gene family.Although the intron numbers and sizes of MdHsf genes were relatively divergent,the conserved motifs and domains of MdHsf proteins were highly conserved because of functional constraints.Based on EST data,72% of the 50 genes (except 14 genes such as MdHsfA2a and MdHsfA3a/b/c)had transcription activities.Selective pressure signatures demonstrated that no positive selection site was identified in the cleaned codon alignments for 36 MdHsf genes based on site-specific model,suggesting that this protein family was controlled by purifying selection.However,branch-site model had identified a total of five positively selected sites in the d and e clade of the phylogenetic tree,i.e.28R,30L,35D,51M and 67V.28R and 30L were included in the Hsf domains,while 35D,51M and 67V were not mapped on the region of Hsf domains,suggesting that purifying selection was the main evolutionary dynamics of functional conservation Hsf domains except for 28R and 30L.In conclusion,various Hsfs existed in apple genome,and the conserved motifs and functional domains were conserved.The majority of them had transcription activity,and the evolution of this family was dominated by purifying selection.

To obtain the transcription factor related to stress conditions in apple rootstock,an apple SBP gene with the full-length cDNA was cloned in Qingzhen 1(apomictic crabapple Pingyitiancha( Malus hupehensis Rehd.)× columnar apple strain CO(Malus × domestica Borkh.) and M26 via homology-based cloning method.The gene cloned was named as MdSBP20 .Bioinformatics analysis to the cDNA showed that the full-length cDNA was 1 362 bp,it's open reading frame possessed 1 362 bp,and encoded 454 amino acids,which had obviously SBP -domain and two zinc finger structures(Zn-1,Zn-2) and the bothway nuclear location signal(NLS).Phylogenetic analysis suggested that MdSBP20 gene had the closer relationship with Pyrus bretschneideri (XM_009339726.1)and Malus domestica (XM_008376435.1).Real-time PCR analysis showed that MdSBP20 gene played a role in response to low temperature,drought,and salt tolerance.It was inferred that QZ1 might has higher resistance than M26 no matter in stress of low temperature,drought,and salt.Based on the results in this experiment, MdSBP20 gene had the significantly effects in apple rootstock response to abiotic stress.

In this study,we aimed to identify the differences of tree morphology,accumulation and distribution characteristics of mineral nutrient in root system of Gala apple young tree with different dwarfing interstocks.The results were expected to reveal the relations between the tree dwarfing ability and accumulation,distribution of mineral nutrient in root system.Gala apple saplings (in pots) grafted onto dwarfing interstocks (GM256,Liaozhen No.2,SH1,SH6,SH38,and SH40) with uniform growth vigour were used as the experimental material.After spring shoot growth stopped,plant height and cross-sectional area were measured.The root morphology indexes were analyzed by WINRhizo analysis software.For the measurement of mineral nutrient contents,healthy plants at the same growth stage were removed from the pots,and were dissected and separated into different diameter root system.The contents and accumulations of mineral nutrient were determined.The results showed that plant height and cross-sectional area of Gala apple trees grafted onto dwarfing interstocks were obviously lower than that of the control.The degree of dwarfing of the interstocks followed the order of SH38 > SH1 > SH6 > GM256 > SH40 > Liaozhen No.2.Differences in mineral nutrient contents in different diameter root system of Gala apple young tree among different dwarfing interstocks were obviously different.The mineral nutrient contents in<0.5 mm diameter root system were highest,and the mineral nutrient contents decreased with the increase of root system diameter.The contents of K and Mg of Gala apple root system for SH38 were highest.Root system for SH1 interstocks had the highest Cu,Fe and Zn contents.The accumulations of P,Ca,Fe in all parts of GM256 interstocks root system and that of K,Mg and Zn in>3 mm diameter root system were higher than control and other treatments.The accumulations of K,Mg and Zn in<3 mm diameter root system for Liaozhen No.2 were highest.The mineral nutrient accumulations of SH38,SH1 and SH6 with weak growth vigour were lower than that of Liaozhen No.2,GM256 and CK with strong growth vigour.The optimal interstocks for Gala apple with better dwarfing effect were SH38,SH1 and SH6.In practical application,site condition,soil supplying nutrient capability and nutrient requirement characteristics of different stock-scion combinations should be fully considered,which for realizing the high quality and high efficiency production of apple.

In order to develop novel disease-resistant transgenic plant materials against ASPV,a virus resistant RNAi plant expression vector was constructed by the method of homologous recombination.With the Malus rootstock of M26,genetic transformation was conducted and positive transgenic plantlets were obtained successfully.Firstly,a conserved segment of coat protein gene of ASPV (489 bp) was cloned by PCR from cDNA of the virus-infective leaves of Royal gala.Then the fragment was inserted into the vector pENTR/SD/D by TOPO cloning technology to form the intermediate vector of pEN-ASPV.After this fragment was replaced and connected into the destination vector pHellsgate12 by LR reaction,the RNAi plant expression vector Phe-ASPV was obtained.And the engineering bacteria EH-ASPV for plant transformation was obtained after the constructed RNAi vector was transformed into Agrobacterium strain EHA105 by alternate freezing and thawing method.By the Agrobaterium mediated method,transformation was conducted with the leaf discs of the Malus rootstock of M26.After culture and subculture for antibiotics screening,bacteria elimination and rooting,compact kanamycin resistant plantlets formed from buds were obtained.After total RNA extracted from the well rooted plantlets,RT-PCR test was conducted and the result showed that the gene fragment of 489 bp carried on the RNAi structure had been transformed into the material of M26,which was expressed in plants on the level of RNA.Through this study,novel transgenic materials were obtained successfully,which laid foundation for disease-resistance evaluation by pathogen inoculation and virus-resistant molecular breeding in the near future.

In order to study the molecular characterization and expression patterns of GA20 oxidase gene in apple dwarfing rootstocks,RT-PCR method was used to clone GA20 oxidase gene (GA20ox1) from cDNA of the apple dwarfing rootstocks No.2 and No.36, and the genes and the amino acid sequences, as well as gene expression on rootstocks and the grafted varieties at various times, was analysed, respectively. Results showed that cDNA of GA20ox1 gene coded a 1 179 bp sequence, and derived a protein with 393 amino acids including termination codon, which the predicted protein molecular weight was 44.3 kDa and theoretical isoelectric point was 5.89.The protein contained conserved domain characteristics of GA20ox1 gene family,and phylogenetic analysis showed that the protein sequence homology with SH40 was at 96.9%. Real-time fluorescent quantitative PCR analysis revealed that the GA20ox1 expression of No.36 rootstock was significantly lower than the control (Malus robusta Rehd.) in June and higher than that in July to August, the expression of the varieties grafted on it was significantly lower than control in July. From July to August, GA20ox1 gene expression levels of No.2 rootstock and the grafted varieties were significantly lower than control.

To explore the sequence characteristics of β-glucosidase gene VmGluI from Valsa mali var. mali (Vmm)and analyze its expression pattern in the process of pathogen infection,the cDNA sequence of VmGluI was amplified from the strong pathogenic strain LXS080601 by using RT-PCR technology.The phylogenetic relationship,physicochemical properties and secondary structure of the protein coded by VmGluI were analyzed by bioinformatics software.The expression levels of VmGluI in apple tiwgs(Malus domestica Borkh.Fuji) inoculated by Vmm were analyzed by Real-time quantitative PCR.The results showed that the cDNA sequence of VmGluI from Vmm was obtained and its GenBank accession number was KY646110.The open reading frame of 1 689 bp encoded 562 amino acid residues with a predicted molecular weight of 62.7 kDa.VmGluI shared characteristic conserved domains of glycosyl hydrolase family 1. α -helices and random coils were its main elements of secondary structure of VmGluI,and β-turns were less.Multiple sequence alignment and phylogenetic tree analysis showed that the encoded protein had the closest genetic relationship with β-glucosidase genes from Vmm (KUI68239.1) and V.mali var. pyri (KUI56905.1),and the similarities of amino acid sequences were 98.7% and 87.8%,respectively.However,the relationship between VmGluI and other β-glucosidase genes from non-pathogenic fungi was far away.The Real-time quantitative PCR results showed that VmGluI was significantly up-regulated in Fuji tiwgs after inoculation by Vmm.Thus,the authors speculated that VmGluI could paly an important role in the pathogenic process of Vmm.The expression level of VmGluI began to increase significantly in detached twigs at 48 h and the highest expression level was 6.3 times higher than that in the control at 72 h.However,the gene expression level increased significantly at 12 h in living twigs,which was 11.2 times higher than that in the control at 96 h.Although there was a low level of gene expression at 48 h,the amount of gene expression was 16.8 times higher than that in the control at 96 h.The results indicated that expression levels and the time course changes of VmGluI were obvious difference in detached and living twigs inoculated with Vmm.

To explore the physiological response of Malus sieversii to salinity stress, Malus sieversii seedlings were cheared with six salinity levels (0,0.2%,0.4%,0.6%,0.8% and 1.0% NaCl) for 4 hours and seven time levels (0,4,8,12,16,20 and 24 hours) in 0.4% NaCl.Contents of proline,MDA,chlorophyll,and soluble sugar in leaves and activity of SOD and POD were mensurated.The results showed that proline content got the peak when NaCl concentration was 0.6% or 12 h in 0.4% NaCl,increasing 63.1% and 27.0% compared to control respectively.Increasing salt concentration and time resulted in higher levels for malondialdehyde content.Chlorophyll content was decreased first,subseguently increased and decreased once more.Among all treatment they were lower than control.Soluble sugar content and the activity of SOD and POD were increased first,then decreased.The results indicated that Malus sieversii could reduce the damage to the plant by accumulating more proline and soluble sugar and increasing the activity of SOD and POD.It was adapted to salinity stress to some extent.

In order to identify the function of MdCaM gene in apple under different abiotic stresses. MdCaM gene was cloned by RT-PCR from apple, and phylogenetic tree was clustered based on amino acid of it. Also, the relative expression levels of MdCaM gene was analyzed by Real-time quantitative PCR (qPCR) method under wound, low-oxygen, high-temperature stress during apple fruit storage. The results showed that Calmodulin gene had high homology in different species, and MdCaM gene had a closer relationship with Arabidopsis thaliana CaM₁. qPCR analysis showed that the transcription level of MdCaM was up-regulated in the early stage, then returned to the normal level after wound and low-oxygen (0 O₂ and 3%O₂) stress, the difference was wound stress peak at 6 h, while low-oxygen (0 O₂) stress peak at 1 h. Also, MdCaM was up-regulated under high-temperature (20℃ and 40℃) stress compared to control,and the expression level was the highest at 12 h under stress,which indicated that MdCaM gene might be involved in the whole process of high-temperature stress-responsive. These results suggested that MdCaM might function as an stress-responsive gene under wound,high-temperature,low-oxygen stresses during apple storage. This research provided a theoretical basis for further study of the function and molecular regulation mechanism of MdCaM.

As the basic component of plant microtubules,tubulins are of great significance to the formation of cytoskeleton. To investigate the effect of one member of the β-tubulin gene family with the transcript number of MDP0000749824.1 on the stem growth,the coding sequence of this gene was cloned from the apple young shoot tips of standard apple variety Yanfu 3 and the columnar variety Telemon,respectively. This sequence was 1 332 bp in length which encoded a peptide with 443 amino acids. The results of DNAMAN alignment showed that the coding sequence of the gene was identical between Yanfu 3 and Telemon.Using DNA of Yanfu 3 and Telemon as templates,1 661 bp nucleotide sequences was obtained by PCR amplification with promoter specific primers. There were six bases differences between them. Promoter sequence analysis revealed that some hormone responsive elements of gibberellin,abscisic acid,methyl jasmonate and salicylic acid existed in it. Cam35S-MDP0000749824-GFP recombinant vector was constructed. Subcellular localization analysis with tobacco leaves indicated that the protein encoded by MDP0000749824.1 was on the cell membrane and microtubules. qRT-PCR detected that the transcript level of this gene in apical stems of columnar apple Telemon was significantly lower than that of standard apple Yanfu 3. It was speculated that the gene could affect the stem elongation be cause its expression level was regulated by hormones.