Study On Tocopherol Content Determination Of Kernel And JrMPBQ Gene Cloning In Walnut(juglans Regia L.)

Vitamin E is an essential fat-soluble vitamin for human body and walnut kernel is an important source of natural vitamin E.The study on four varieties of Juglans regia vitamin E content of nuts is determined to measure and analyze.To Xiangling walnut embryo tissue with the method using homologous sequences cloned gene sequence jrMPBQ.The main results are followed.1. The Analysis for Tocopherol Content of Kernel in JuglansFourteen samples were chosen as study materials including ten varieties of Juglans regia(four varieties and six F1 hybrids of Yuan Lin×Qinglin), two varieties of J.nigra, one sample of J.microcarpa and one sample of J.cathayensis. The HPLC technique were used to determine the content ofα,β,γ,δ-tocopherol and the correlationship among the content of tocopherol with the nut traits were analyzed. The results showed that the differences of tocopherol contents among the four varieties of Juglans regia were very significant in the year of 2008 and 2009 respectively. The total tocopherol content of four varieties of Juglans regia were ranging from 8.51±0.09 to 35.28±1.23mg/100g . The total tocopherol content of J.nigra was the highest in the year of 2008(20.21±0.54 mg/100g),but J.cathayensis was the highest in the year of 2009(35.28±1.23mg/100g);γ-Tocopherol was the major compound in all samples, ranging from 6.59±0.14 to 33.19±1.12mg/100g,followed byδ- andα-tocopherols, ranging from 0.75±0.03 to 2.51±0.01mg/100g and from 0.27±0.02 to 1.53±0.71mg/100g ,respectively.β- Tocopherol was the lowest , ranging from 0.00±0.00 to 0.20±0.01 mg/100g. The total tocopherol mean value was 8.51±0.09 mg/100g in 2008 and 13.11±0.17 mg/100g in the year of 2009 of J.nigra,which was lower than others significantly at P<0.01.γ-Tocopherol and total tocopherol content was significantly and positively correlated (r = 0.981, P﹤0.01), the rate of walnut kernel yield with tocopherol contents was positively correlated( r = 0.553, P﹤0.05). In four varieties and six F1 hybrids of Yuan Lin×Qinglin from J. regia ,the total tocopherol content were ranging from5.57±0.20 to 18.99±0.45mg/100g.γ-Tocopherol was ranging from 3.83±0.13 to 14.88±0.40mg/100g.δ- andα-tocopherols were ranging from 0.67±0.15 to 3.59±0.26mg/100g and from 0.09±0.01 to 0.95±0.05mg/100g ,respectively.The content ofβ- Tocopherol was relatively low , ranging from 0.00±0.00 to 0.33±0.01mg/100g. The analysis also showed that the differences of tocopherol contents among the ten samples of J. regia were also very significant in two years by t test.There was transgressive inheritance in the tocopherol contents of six hybrids from Yuan Lin×Qinglin. 2. Cloning of jrMPBQ geneTotal RNA was extracted from nut embryo tissue Xiangling walnut by CTAB method in the active nutrition metsbolism period.The primer was designed according to sequence of homologous gene of conserved region and jrMPBQ gene was cloned by RT-PCR through 3′RACE and 5′RACE. jrMPBQ gene is 1280bp, encoding a polypeptide containing 340 amino acid sequence.Code in both DNA sequence and peptide sequence have relative higher homology with code in MPBQ/MSBQ sequence of Populus trichocarpa, MPBQMT sequence of Hevea brasiliensis and MPBQ/MSBQ sequence of Lactuca sativa.This polypeptide contained 10 Serine,1 Threonine and 4 Tryptophan loci and it might be site of protein kinase phosphorylation.The minimum of its hydrophobicity is -2.378,and the maximum of its hydrophobicity is 2.556. Extended strand accounted for 45.00%,and Alpha helix accounted for 39.41%, Random coil accounted for 15.59% in the secondary structure.MPBQ is belong to methyltransferase family in the function,which catalyzed by 2- methyl- 6- phytyl- 1,4- benzoquinol(2M6PBQ) to 2,3-dimethyl-5-phytyl- benzoquinol(2,3DM5PBQ) in vitamin E synthesis, which isγ- tocopherol andα-tocopherol biosynthesis rate-limiting step.