| Oil-tea Camellia(Camellia oleifera Abel.), one of the edible oil woody species endemic to China, is widely distributed in mountainous and hilly areas of southern China. The species grows in acid red-yellow soil with very low phosphorus content. Phosphorus(P) is one of the necessary macronutrients for plant growth, and P deficiency in soil is a key factor for limiting the growth and yield of oil-tea Camellia. However, Oil-tea Camellia have formed a series of complex adaptive mechanism to low P in the process of evolution, such as some root morphological, physiological and biochemical adaptations. Therefore, we can screen out the P-efficiency varieties by making full use of the genetic resources diversity of oil-tea Camellia, and then excavate and study the genes related to P utilization efficiency(PUE). Furthurmore, we can improve PUE by the marker-assisted selection breeding and genetic engineering breeding. It will become an effective way to solve P deficiency in the soil. Here, two oil-tea Camellia clones cutting seedlings were used as experimental materials, and the two clones have significant differences in PUE. We performed a comparative analysis of physiological indexes of the two clones under low phosphorus stress, and screened out the relevant indicators of PUE. Besides, the relevant genes of PUE in oil-tea Camellia were dug out by transcriptome sequencing and real-time quantitative PCR(RT-qPCR) analysis in the two clones under different phosphorus levels The main findings are described as followings:1.The relevant indicators of PUE were screened out by comparative analysis of physiological and biochemical indexes of the two clones under low P stress.(1) Phosphorus deficiency was significantly decreased the height, biomass(root, stem,leaf and whole plant), root parameters(total root length, surface area and volume) and P uptake efficiency(accumulation of P) of the two clones, while it markedly increased root-shoot ratio and PUE. The height, plant biomass, root parametersand P utilization efficiency in ‘changlin 166’ were higher than those in ‘changlin 4’ under the low phosphorus level, which indicated ‘changlin 166’ was a high PUE genotype clone.(2) Phosphorus deficiency was significantly increased acid phosphatase activity(roots, root exudates and leaf), CS, NAD-MDH and PEPEC enzyme activity of roots and anti-oxidation protective enzymes activities(SOD, POD and CAT) of roots in the two clones. Besides, the IAA, GA and ABA contents were also significantly increased while ZR was obviously reduced. The acid phosphatase activities, CS, NAD-MDH and PEPC activity, anti-oxidation protective enzymes(SOD, POD) activities and endogenous hormones(IAA, GA, ABA and ZR) content in ‘changlin 166’ were higher than those in ‘changlin 4’ under phosphorus deficiency.(3) Low P stress was significantly decreased chlorophyll, photosynthetic gas exchange parameters(Pn, Tr, Gs and Ci), chlorophyll fluorescence parameters(Fv/Fm, Y(II), qL and ETR) in the two clones, and increased chlorophyll fluorescence parameters(Y(NPQ), Y(NO) and qN). The chlorophyll, Pn, Tr, Gs, Ci, Fv / Fm, Y(II), q L and ETR value of ‘changlin 166’ were higher than those in ‘changlin 4’ under P deficiency, while Y(NPQ), Y(NO) and qN were lower ‘changlin 4’, indicating that ‘changlin166’ suffered less damage than ‘changlin 4’ under low phosphorus stress, and photosynthetic efficiency of ‘changlin 166’ was higher than that in ‘changlin 4’.(4) According to the relevant results of physiological indicators, we regarded root dry weight, total biomass, root-shoot ratio as key indicators for evaluating the P-efficiency genotype of oil-tea Camellia; PUE was selected as direct indicators to make sure that it was a P-efficiency genotype of oil-tea Camellia; photosynthetic parameters and chlorophyll fluorescence parameter were used as a secondary indicator for testing the low phosphorus tolerance of clones in the early stage.2. Transcriptome analysis of roots in two Camellia clones root under different phosphorus levels. A total of 614,364,144 clean reads were obtained by transcriptome sequencing, with Q20 more than 97%. A total of 208,506 unigenes were gained by assembling with an average length of 1452 bp of N50. According to 166LP-VS-166 HP and 166LP-VS-4LP differentially expressed genes and function of related genes, a total of 16 genes were screened out for following research, which is including phosphate transporter protein family, acid phosphatase gene family, organic acid secretion related genes(CS, NAD-MDH, PEPC and ALMT), transcription factor PHR2 and lateral root associated gene PDR2. A total of 16 genes were screened out and they may contribute to the P-efficiency of ‘changlin 166’.3. The response of the genes related to phosphorus efficiency under low phosphorus and expression analysis of different clones under different P levels. PHR2, PDR2, CS, ALMT, Pht1,9, Pho1-3 and PAP1 genes were dug out as key genes which may contribute to the P-efficiency of ‘changlin 166’.4. Cloning and bioinformatic analysis of PHO1-3, ALMT, CS and PHR2 gene. CoPHO1-3, CoALMT, CoCS and CoPHR2 were isolated from the roots of Camellia oleifera by RT-PCR. Their full-length cDNAs were 2406 bp, 1761 bp, 1416 bp and 1419 bp, respectively; they encoded predicted proteins of 801, 586, 471 and 472 amino acids. Phylogenetic tree analysis suggested that CoPHO1-3 have close genetic relationship with Sesamum indicum、Glycine soja and Glycine max. The other three genes CoALMT、CoCS and CoPHR2 had close genetic relationships with Vitis vinifera.5. CoCS and CoPHR2 were transformed into Arabidopsis thaliana. The plant expression vector of CoCS and CoPHR2 were constructed by Gateway technology, and were transformed into Arabidopsis thaliana by the floral dip method. By hygromycin resistance screening and PCR analysis, 6 and 5 positive transgenic lines(containing CoCS) were identified from the Arabidopsis mutant cs and wild-type; 8 and 6 positive transgenic lines(containing CoPHR2) were idengtified from Arabidopsis mutant phr1 and wild-type WT. |
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