| Potato tubers, a modified subterraneous stems, are originated from the stolon apical cell. Researches on tuber development are focused on morphogenesis and their related characters. Because they are critical to improve potato yield and qualities, tuber formation are influenced by many factors, such as photoperiod, hormones, temperature and sucrose. However, factors that act on tuber shape are still unclear. In this research, we analysis the flanking sequences of a long-tuber-shape mutant. It revealed that a special gene may play a role in tuber shape. The result will promote the study on tuber shape. We also investigated the relationship among five phytochromes (PHYA、PHYB、 PHYB2、PHYE and PHYF) and microtuber formation. This work can offer references for a better understanding of the role of phytochrome on potato microtuber formation. The main results are as follows.Flanking sequences analysis of the long-tuber-shape insertion mutantpCL2b-2is a long-tuber-shape mutant, which was obtained from potato transgene lines carrying a T-DNA insertion. First, we verified the phonotype of this mutant. The results showed that the length-width ratio of microtubers of the mutant in vitro (1.78±0.15) was significantly greater than that of the wild type (1.33±0.05)(p<0.05). The shape of normal tubers grown in pots was also significantly different between pCL2b-2and the wild type potato E3(length-width ratio1.50±0.06vs.1.29±0.06,p<0.05). We got a965bp sequence by TAIL-PCR. Among them,139bp belonged to RB, while the rest826bp were potato genome sequence. It showed89%similarity to the4th intron of isopentenyltransferasegene (IPT) by blasting the826bp sequences in PGSC (http://potatogenome.net/index.php/Main_Page).It suggested that the T-DNA inserted into the IPT. Further analysis demonstrated that the T-DNA replaced317bp of the4th intron. IPT was the key gene for cytokinin synthesis. The IPT gene cloned from potato has not been reported. Here, we named it StIPT. The insertion of this gene changed the tuber shape, which suggested that StIPT might participated in the potato tuber morphogenesis. The effect of phytochromes on potato microtuber formation.We found five phytochrome genes in PGSC. PHYA and PHYB were same as the previous reports. According to their homologous genes in other species, the other three phytochromes were named PHYB2, PHYE and PHYF. To analysis the responses of the5phytochromes to photoperiod, the E3was cultivated in LD (16h/d) in vitro for3weeks and then transferred to LD and SD (8h/d) respectively. In two days, we collected the materials every4hours to analysis the expression levels of the5phytochromes. The results showed that4phytochromes presented different circadian rhythms except PHYB. PHYA reached its peak during dark in SD conditions, PHYB2emerged an up-regulated expression in dark and a down-regulated expression in light after12hours in SD. PHYE and PHYF expressed similar in either LD or SD. And PHYE displayed clear circadian rhythms, which increased to peak in4hours in light and then decreased and maintained at a low level in dark. PHYF showed a time rhythms with2peaks per24hours.To understand the effect of these five phytochromes on the potato microtuber formation, we constructed five RNAi vectors of each phytochrome and transformed to E3by agrobacterium mediated transgenic technology respectively. All151positive transgenic lines were obtained and the highest RNAi efficiency was98%. Three to five positive lines of each gene were selected to detect tuberization ratio. The result displayed that PHYB and PHYF RNAi lines got higher tuberization ability than E3in LD. It indicated PHYB and PHYF may play important roles in potato formation controlled by photoperiod. We analysised the tuber development related genes StCO and StSP6A in multiple RNAi lines. StCO showed no regular changes, while StSP6A in all of the PHYB and PHYF RNAi lines demonstrated significant increase (p<0.05) in LD. It is suggested that PHYB and PHYF might participate in the tuber formation by regulating StSP6A. In this study, it is first reported that PHYF is an important effector in tuber formation. |
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