Identification of Rubisco rbcL and rbcS in Camellia oleifera and their potential as molecular markers for selection of high tea oil cultivars

Abstract

Tea oil derived from seeds of Camellia oleifera Abel. is high-quality edible oil in China. This study isolated full-length cDNAs of Rubisco subunits rbcL and rbcS from C. oleifera. The rbcL has 1,522 bp with a 1,425 bp coding region, encoding 475 amino acids; and the rbcS has 615 bp containing a 528 bp coding region, encoding 176 amino acids. The expression level of the two genes, designated as Co-rbcL and Co-rbcS, was determined in three C. oleifera cultivars: Hengchong 89, Xianglin 1, and Xianglin 14 whose annual oil yields were 546.9, 591.4, and 657.7 kg ha(-1), respectively. The Co-rbcL expression in 'Xianglin 14' was significantly higher than 'Xianglin 1', and 'Xianglin 1' was greater than 'Hengchong 89'. The expression levels of Co-rbcS in 'Xianglin 1' and 'Xianglin 14' were similar but were significantly greater than in 'Hengchong 89'. The net photosynthetic rate of 'Xianglin 14' was significantly higher than 'Xianglin 1', and 'Xianglin 1' was higher than 'Hengchong 89'. Pearson's correlation analysis showed that seed yields and oil yields were highly correlated with the expression level of Co-rbcL at P < 0.001 level; and the expression of Co-rbcS was correlated with oil yield at P < 0.01 level. Net photosynthetic rate was also correlated with oil yields and seed yields at P < 0.001 and P < 0.01 levels, respectively. Our results suggest that Co-rbcS and Co-rbcL in particular could potentially be molecular markers for early selection of high oil yield cultivars. In combination with the measurement of net photosynthetic rates, the early identification of potential high oil production cultivars would significantly shorten plant breeding time and increase breeding efficiency.

Keywords: Camellia oleifera; Rubisco; camellia oil; photosynthesis; rbcL; rbcS; tea oil.

FIGURE 1

Camellia oleifera “Xianglin 14’ produced abundant fruit. Fruit diameters were up to 4 cm (A); seed appearance at seed shell opening (B); a mix of seeds, nutlets, and fruit (C); and tea oil derived from seeds (D).

FIGURE 2

Mean dry seed (A) and oil (B) yields of three cultivars Hengchong 89, Xianglin 1, and Xianglin 14 grown at the Experimental Station of the National Engineering and Technology Research Center of Oil-Tea Camellia, Changsha, Hunan, China from 2011 to 2013. Bars represent standard error with n = 9. Different letters above the bar represent significant difference at P < 0.05 level by Duncan’s Multiple Range Test.

FIGURE 3

Polymerase chain reaction cloning of rbcL from leaves of C. oleifera and its recombinant strains. (A) Lane 1: the PCR amplification fragment of rbcL with an expected band at 1,522 bp for rbcL amplified by PCR. (B) Lanes 1 and 2 represent the positive recombinant strains. M represents 100 bp plus DNA ladder.

FIGURE 4

Polymerase chain reaction cloning of rbcS from leaves of C. oleifera and its recombinant strains. (A) The degenerate PCR amplification fragment of rbcS in lane 1. (B) Lanes 1, 2, and 3 are positive recombinants of rbcS. (C) Lane 1 indicates the 5′ RACER result of Co-rbcS. (D) Lanes 1–6 were positive recombinants of the 5′ RACER of Co-rbcS. (E) Lane 1 is the 3′ RACER result of Co-rbcS. (F) Lanes 1–10 are positive clones of Co-rbcS. M represents 100 bp plus DNA ladder.

FIGURE 5

Comparison of the deduced amino acid sequences of the rbcL protein from Camellia oleifera, Camellia sinensis, Camellia taliensis, Prunus persica, Pyrus pyrifolia, pine (Pinus thunbergii), and tobacco (Nicotiana tabacum). Amino acid residues identical to those of C. oleifera rbcL protein are indicated by dots. Hyphens indicate gaps. The arrow indicates the possible processing site of mature protein in C. oleifera; and active site residues are overlined and boxed.

FIGURE 6

Alignment of the amino acid sequence of rbcS from Camellia oleifera with the sequence from Camellia sinensis, Gossypium hirsutum, Pyrus pyrifolia, Brassica napus, and Arabidopsis thaliana. A. thaliana 1B, 2B, and 3B represent A. thaliana ribulose bisphosphate carboxylase small chain 1B (NP_198659), A. thaliana ribulose bisphosphate carboxylase 2B small chain (NP_198658), and A. thaliana small chain 3B (NP_198657), respectively. Amino acid residues identical to those of C. oleifera rbcS protein are indicated by dots. Hyphens indicate gaps. The arrows indicate the possible processing site of mature protein in C. oleifera.

FIGURE 7

Expression of Co-rbcL (A) and Co-rbcS (B) in leaves of three cultivars Hengchong 89, Xianglin 1, and Xianglin 14 analyzed by real-time quantitative PCR, and the expression levels were normalized based on internal control of GAPDH gene. Bars represent standard error with n = 3. Different letters above the bar represent significant difference at P < 0.05 level by Duncan’s Multiple Range Test.