Genome-wide identification and expression analysis of the SWEET gene family in daylily (Hemerocallis fulva) and functional analysis of HfSWEET17 in response to cold stress.

BACKGROUND: The Sugars Will Eventually be Exported Transporters (SWEETs) are a newly discovered family of sugar transporters whose members exist in a variety of organisms and are highly conserved. SWEETs have been reported to be involved in the growth and development of many plants, but little is known about SWEETs in daylily (Hemerocallis fulva), an important perennial ornamental flower. RESULTS: In this study, 19 daylily SWEETs were identified and named based on their homologous genes in Arabidopsis and rice. Phylogenetic analysis classified these HfSWEETs into four clades (Clades I to IV). The conserved motifs and gene structures showed that the HfSWEETs were very conservative during evolution. Chromosomal localization and synteny analysis found that HfSWEETs were unevenly distributed on 11 chromosomes, and there were five pairs of segmentally duplicated events and one pair of tandem duplication events. The expression patterns of the 19 HfSWEETs showed that the expression patterns of most HfSWEETs in different tissues were related to corresponding clades, and most HfSWEETs were up-regulated under low temperatures. Furthermore, HfSWEET17 was overexpressed in tobacco, and the cold resistance of transgenic plants was much higher than that of wild-type tobacco. CONCLUSION: This study identified the SWEET gene family in daylily at the genome-wide level. Most of the 19 HfSWEETs were expressed differently in different tissues and under low temperatures. Overexpression further suggests that HfSWEET17 participates in daylily low-temperature response. The results of this study provide a basis for further functional analysis of the SWEET family in daylily.

[Optimizing of cDNA preparation for next generation sequencing].

Next generation sequencing has already been used for genomic analysis of microorganis, human being, animals, and plants. Sample preparation is prerequisite and most important for large-scale sequencing. There are two major interferences for large-scale sequencing, polyA and abundant genes' concealment for rare genes. In order to solve these problems, we used total RNA extracted from violaceae leaves to produce double stranded cDNA. DSN nuclease was used to treat the ds cDNA prior to removing the polyA. Randomly sequencing 100 clones of the treated cDNA showed that there were 94 independent clones in the treated sample, and the sequences did not contained polyA. However, only 62 independent clones were found in the untreated sample, and 15 of the sequencing files were affected by polyA. By randomly sequencing of the treated cDNA, we also found two clones encoded two interested genes. We failed to isolate these genes although the protein mass peaks of them had been found in the MALDI-TOF trace. Furthermore, we designed primers from two known genes with different expression abundances. The PCR yields were approaching similar using the treated cDNAs as templates. These results showed that, removal of the polyA and enrichment of rare genes with DSN can meet the requirements of large-scale sequencing and discovery of new genes.

[Carbohydrate metabolism during fruit development of bayberry (Myrica rubra Sieb. et Zucc.)].

The dynamics of dry and fresh weight, the glucose, fructose, sucrose, titratable acid contents, and activities of sucrose-metabolizing and hexose-metabolizing enzymes were examined in developing fruits of bayberry (Myrica rubra Sieb. et Zucc. cvs. 'Wuzi' and 'Biqi'). The results showed the dry and fresh weight of bayberry fruit increased with fruit development and maturation (Fig. 1), with the highest increase rate of dry matters and water occurring during later stage of fruit development (about 10 d before maturation). The change in titratable acid followed a course of "low-high-low" in developing bayberry fruits (Fig. 3). The titratable acid content reached its peak at about 18 d before fruit maturation, and then decreased rapidly. The sugar compositions in fruits of bayberry cv. 'Wuzi' were different from those in fruits of bayberry cv. 'Biqi'. The main sugar accumulated in fruits of bayberry cv. 'Wuzi' was sucrose, accounting for 2/3 of total sugars but the sucrose content in fruits of bayberry cv. 'Biqi' was below 50% of total sugars. The fructose content in fruits of bayberry cv. 'Wuzi' was 4% higher, but that in fruits of bayberry cv. 'Biqi' was 12% lower than glucose content (Fig. 2). The activities of sucrose cleavage enzymes (invertase and cleavage activity of SS) in the fruit of bayberry cv. 'Biqi' increased with fruit development and maturation, but those activities in fruit bayberry cv. 'Wuzi' were almost stable during fruit development with lower levels of enzyme activities in fruit of cv. 'Wuzi' than in cv. 'Biqi' throughout fruit development (Fig. 4 and Fig. 5A). The SPS activity increased during fruit development (Fig. 6), however, the activity peak of synthetic activity of SS occurred at the middle stage of fruit development (Fig. 5B). The FRK activity in fruit of bayberry cv. 'Wuzi' was higher than that of HXK, but the reverse was in fruit of bayberry cv. 'Biqi' (Fig. 7). These results suggested that the 2-3 weeks before fruit maturation was a key phase for the bayberry development and the formation of fruit quality. There was a correlation between water transport and dry matter accumulation. The different sucrose constitutions between two varieties may be attributed to the differences in the activity levels of the sucrose cleavage enzymes while the difference in the ratio of glucose content to fructose content may be caused by the different activity levels of the hexose-metabolizing enzymes.

[Genetic transformation of peach immature cotyledons with its antisenes ACO gene].

Genetic transformation of peach immature cotyledons with its ACO antisense gene was studied by using particle bombardment method through Agrobacterium tumefaciens. Kanr shoots and Kanr plantlet were obtained. The plantet with ACO antisenes gene through Agrobacterium tumefaciens was obtained by micrografting technique and survived for nearly one month. The results of the PCR, PCR-southern, genomes southern hybridization analysis and GUS color reaction of some Kanr materials showed in some degree that the peach ACO antisens gene was integrated into peach genomes.

[The relationship of fructokinase and sugar accumulation during fruit development in satsuma mandarin].

Sugars accumulation and fructokinase activity during satsuma mandarin fruit development in relation to the effect of extra nitrogenous fertilizer on the activity and expression of fructokinase were studied. The results exhibited that fructokinase activity in the tissues of edible and peel decreased during fruit development, which coincided with the accumulation of sugars, while the contents of sucrose and glucose decreased, and the activity of the enzyme increased in peel tissues of ripened fruit. After fertilizing with extra urea, the ratios of sucrose and fructose decreased in ripe fruit, while that of glucose increased compared to the control. The activity of fructokinase presented on a protein basis increased in treated fruit. Northern analysis confirmed that extra nitrogenous fertilizer enhanced the expression of Cufrk1 at the late stage of fruit development, but had no effect on Cufrk2. The results suggest that the two different genes of citrus FRK may play distinct roles in sink metabolism and Cufrk1-encoded fructokinase protein could be induced by fertilization with extra nitrogen.