Identification Of Cold Tolerance Germplasm And The Cold Tolerance Mechanism In Loquat

Loquat(Eriobotrya japonica Lindl.),an evergreen subtropic fruit tree,originated in China.It is a popular fruit tree because the fruit ripens at the beginning of summer and unique flavor,rich nutrients,highly medical and ornamental value.The white-fleshed loquat has better fresh quality than yellow-fleshed loquat.It is the most important fruit tree in Jiangsu province.Loquat usually blossoms in autumn and winter,the coldest season of a year,flowers and young fruits are vulnerable to freezing and result in yield lost.Low temperature freezing injury in winter is the most important limiting factor for loquat production.Jiangsu Province is the northern edge of loquat economic cultivation.There are serious or slight freezing injuries in winter every year.The cold resistant of 25 white-fleshed loquats was evaluated with the REC mathed and the logistic equation,one cultivar was selected for subsequent experiments.Based on the analyse of physiology,transcriptome,gene function and proteome,the cold tolerance mechanism of white-fleshed loquats was further studied.The main findings are as follows:1.Using the leaves of 'Baiyu' loquat as materials,the cold tolerance test system of white loquat was established by REC mathed and the logistic equation,then 25 white loquats were determined.The results showed that the relative conductivity(REC)changed as 'S' curve when the cold treatment time over 8 h,and the semi-lethal temperature(LT50)was calculated by the logistic equation.The LT50 result showed 'Guanyu' has the most cold-resistant with-15.95? LT50,followed by‘American species','Copper skin',‘Shanghai species','Baiyu'and 'Meiyu' and other white loquat resources.2.The changes of SP,SS and MDA contents in 'Guanyu' leaves were determented under different low temperature stress and the changes of Pro,PAL and An contents within 24 h under 4? were studied.The results showed the SP content followed a fluctuating tendence with the decrease of low temperature wihle SS and MDA content increased with the decrease temperature.This indicated plants could resist low temperature by increasing MDA and synthesis of SS.Pro,PAL and AN showed the same trend,increased before 12h and decreased after 24h under 4?.Therefore,it indicates that the osmotic regulators Pro and the antioxidants PAL and AN take participate in the process of cold resistant.3.RNA-Seq high-throughput sequencing was used to analyze the transcriptome of'Guanyu' under 4?.The results showed that 122,081 Unigenes,61,357 up-regulated genes and 60,724 down-regulated genes.101,013 nigene were annotated in public databases such as NR,NT and Swiss-Prot.88,120 Unigenes were annotated in the NR database,53.8%highly matched the Amygdalus persica sequence;38,604 Unigenes had specific functional definitions in the COG database,involving 25 functional classifications;60,464 Unigenes had specific functional definitions in the GO,divided into three functional classifications;55,138 Unigenes can be classified into 128 biological pathways.The result of differential gene expression analysis showed there were 1,210 DEGs,599 up-regulated DEGs,and 611 down-regulated DEGs.15 of up-regulated DEGs were specifically up-regulated and the expression of 28 down-regulated DEGs decreased to zero.The top 50 DEGs were up-regulated,mainly COL,AP2 and MYB transcription factors.The top 50 DEGs were down-regulated,but the transcription factors were absent.Most of the genes involved in carbohydrate metabolism,signal transduction,RNA transport and so on were in the whole metabolic pathway.KEGG functional analysis showed that 765 DEGs genes with pathway annotations were annotated in 106 pathways,7 of which were significantly enriched.The main metabolic pathways involved were circadian rhythmic plant,flavonoid and flavonol biosynthesis,flavonoid biosynthesis,photosynthesis-antenna protein,Diphenylethylene glycol biosynthesis.The biosynthesis of olefins and Zingiber,the degradation of limonene and pinene,and the signal transduction of plant hormones.4.iTRAQ method was used to isolate and identify the protome of‘Guanyu'under 4?for 0h,4h,and 8h.The results showed that 22765 peptides were obtained and 4582 proteins were identified.GO,COG and KEGG libraries were selected for functional annotation.4307 proteins were annotated in GO library and belonged to 46 terms.3038 proteins were annotated in COG library and belonged to 24 terms.2705 proteins were annotated in KEGG library and belonged to 261 pathways.Differential protein analysis showed that there were only 33 proteins,7.43%of the 444 proteins,shared by 0h,4h,8h under 4?.Compared with 0 h,there were 300 DEPs at 4 h,179 up-regulated and 121 down-regulated;Compared with 0 h,there were 97 DEPs at 4 h,91 up-regulated and 6 down-regulated at 8 h.Compared with 4h,there were 318 DEPs at 8 h,192 up-regulated and 126 down-regulated.Pathway enrichment analysis showed that there were six common KEGG pathways in PD4:PD0 and PD8:PD4,including antigen treatment and presentation,nitrogen metabolism,glutathione metabolism,aminoglycose and nucleotide metabolism,pentose and glucose conversion,and tetracycline biosynthesis pathways.The comparative analysis of transcriptome and proteome showed that only 27 genes were shared DEGs and DEPs.Most of the high-expression genes could not be found in DEPs.The correlation between transcriptome and proteome was very low.Both transcriptome and proteome analysis and qRT-PCR indicated that S6PDH,ANS and PAL might play an important role in the cold response of loquat.5.Based on the transcriptome sequencing,RACE was used to clone the full length of EjCBF1,EjCBF3 and EjCBF4.The ORF of EjCBF3,711bp,encodes 236 AA while EjCBF4,789bp and 262 AA.EjCBF3 and EjCBF4 contain AP2 domain,one nuclear localization signal domain and one DSAWR characteristic sequence at the side of AP2 domain.The result of blast online showed that EjCBF3 and EjCBF4 had high homology with apple and pear CBF genes in nucleic acid and amino acid level,but poor homology with Arabidopsis.The realtime RT PCR of EjCBF3 and EjCBF4 showed the expression of them had same pattern under 4?,increased then decreased.During 0.5h to 1h the expression increased quickly and keep at high level from 4 h to 12 h.The highest expression of EjCBF4 was at 10 h and the EjCBF3 at 12 h.