| Loquat (Eriobotrya japonica L) is an excellent and characteristic famous subtropical fruits native from China. Chilling have become a restricting factor and bottleneck to be solved immediately for development of loquat industry.Chilling tolerance was explored systematically by some techniques such as scanning electron microscope and transmission electron microscopy, potassium pyroantimonate precipitation and electron cells chemistry, liquid chromatography, mRNA differential display and rapid amplification of cDNA ends and so on in different types of loquat under temperature conditions artificially controlled for developing technological systems to identify chilling tolerance of loquat with leaves replacing fruits, and enriching theories on chilling-tolerant mechanism in loquat, which would lay foundation for further research on molecular mechanism of chilling tolerance and provide value references for chilling-tolerant breeding of other fruits. The main results were as follows:1. Anatomical structures of leaf in some loquat cultivars were observed referring to the results of chilling tolerance in other plants. The results showed that the thickness of leaf, Corneous layer, the epidermis, stockade tissue, sponge tissue were differences among different samples in the same cultivar, but that the ratio of thickness of stockade tissue and sponge tissue, vales of CTR and SR were relatively stable and consistent. It was concluded preliminarily that the strength of chilling tolerance was as follows: wild oak leaf loquat, Krainian bow white, Zaozhong No.6, Taicheng No.4, Xiangzhong No.11; and that the chilling tolerant cultivars were characteristic of small free space between stockade tissue and sponge tissue and of closed mesophyll cells, closed circular fibrovascular bundle in leaf veins.2. The content of MDA and the activities of enzymes removing active oxygen in leaves and fruits of six loquat cultivars were determined for exploring the relationship between some relative physiological indexes in leaves and fruits under stress of low temperature leaves and chilling tolerance of loquat cultivars. The results showed that the content of MDA increased continuously with treating time under low temperature prolonging, that the activities of anti-oxidant enzymes such as POD, SOD, CAT and APX increased only at the earlier stage of tress at low temperature ( treating for 1.5 hours at -3℃). Therefore, the adaptabilities varied in different types of loquat in metabolic activities, the content of MDA were stable in the high chilling tolerant varieties under stress of low temperature, the activities of four enzymes such as POD, SOD, CAT and APX removing active oxygen maintained at a higher level.3. The changes of content of four types of endogenous hormones such as IAA, GA3, ZT and ABA in leaves of three types of loquat (White bow Ukraine, Zaozhong No.6 and wild oak leaf loquat) were determined to exploring the relationship between chilling tolerance of loquat cultivars and endogenous hormones. The results showed that the content of IAA A and GA3 firstly increased, then decreased at the earlier stage of tress under low temperature in Zaozhong No.6 and wild oak leaf loquat which had stronger metabolism by adapting themselves under tress at low temperature for short time, and didn't stop growing and developing at low temperature; that the changes of content of ABA was a double-peak curve, the emerging time of the second peak was roughly same as the time chilling occurred, but the changes of content of ABA in was a single-peak curve Zaozhong No.6. It could be concluded that plant endogenous hormones perhaps acted as signal transduction which mechanism was similar to calcium ions, and their relative physiological and biochemical mechanism could be explicated.4. The changes of level, moving characteristics of calcium ions and cell ultra-structure in leaves of Zaozhong No.6 and wild oak loquat were observed by potassium antimonate precipitation and electron microscopic cyto-chemistry and some relative physiological indexes also were determined for clarifying the mechanism of calcium ions. Low temperature stimulated moving of calcium ions from cell wall and vacuole to cytoplasm, which led to sharp increasing of concentration of calcium ions in cytoplasm. Therefore, buffer of calcium ions was related with chilling tolerance of loquat, and optimum content of calcium ions could increase chilling tolerance of loquat.5. Three positive cDNA fragments such as A1, C1 and C2 related with chilling tolerance of loquat were isolated by combination of mRNA differential display and reverse northern blotting. The nucleotide sequence of A1 was identified with that of histone H4 genes in castor and arabidopsis, shepherd and poplars by more than 80%, it was deduced that A1 perhaps was histone H4 gene. The nucleotide sequence of C1 was identified with that of ATP-dependent Clp protease genes in apple and peach by 91% and 71%, respectively. It was deduced that C1 perhaps was ATP-dependent Clp protease gene. The nucleotide sequence of C2 was identified with that of an unknown protein in poplar only by about 15%, it was deduced that C2 perhaps was a novel gene. Full-length cDNAs of Clp and H4 genes were cloned by RACE, and their bio-functions were predicted by bioinformatic softwares,which would lay foundation for cultivating novel cultivars of loquat with high chilling tolerance by genetic engineering.
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